Origin recognition complex subunit 2


Transcriptional Regulation

One possible mechanism by which E2F transcription factor 2 (E2F2) could inhibit DNA replication is to act as a transcription factor to modulate the expression of at least one crucial replication factor. For example, if E2F2 was part of a repressor complex, loss of E2f2 function could lead to increases in replication gene expression that might trigger widespread DNA synthesis. In order to test this idea, an examination was made of the abundance of several mRNAs encoded by genes either known to be (e.g. Orc1, RNR2, PCNA) or possibly (e.g. Orc2 and Orc5) regulated by E2F. RNA was extracted from total follicle cell preparations and subjected to RT-PCR. Relative to rp49 controls, more Orc5 mRNA was reproducibly (n=4) detected in Df(2L)E2f2329/Df(2L)E2f2329 or Df(2L)E2f2329/E2f1-188 mutant samples, compared with wild type. An increased Orc2 mRNA level was also detected in some experiments (two out of four). For Orc1, RNR2 and PCNA there was no substantial difference in the amount of mRNA detected between wild type and E2f2 mutants. These data suggest (1) that E2F target genes are expressed at or above wild-type levels after loss of E2f2 function and (2) that de-repression of specific target genes, such as those encoding members of the ORC complex, could contribute to the inappropriate DNA synthesis seen in E2f2 mutant follicle cells (Cayirlioglu, 2001).

DNA replication-related element (DRE) and the DRE-binding factor (DREF) play an important role in regulating DNA replication-related genes such as PCNA and DNA polymerase alpha in Drosophila. Overexpression of DREF in developing eye imaginal discs induces ectopic DNA synthesis and apoptosis, which results in rough eyes. To identify genetic interactants with the DREF gene, a screen was carried out for modifiers of the rough eye phenotype. One of the suppressor genes identified was the Drosophila orc2 gene. A search for known transcription factor recognition sites revealed that the orc2 gene contains three DREs, named DRE1 (+14 to +21), DRE2 (-205 to -198), and DRE3 (-709 to -702). Band mobility shift analysis using Kc cell nuclear extracts detected the specific complex formed between DREF and the DRE1 or DRE2. Specific binding of DREF to genomic region containing the DRE1 or DRE2 was further demonstrated by chromatin immunoprecipitation assays, suggesting that these are the genuine complexes formed in vivo. The luciferase assay in Kc cells indicated that the DRE sites in the orc2 promoter are involved in a transcriptional regulation of the orc2 gene. The results, taken together, demonstrate that the orc2 gene is under the control of DREF pathway (Okudaira, 2005).

Targets of Activity

In the yeast Saccharomyces cerevisiae, sequence-specific DNA binding by the origin recognition complex (ORC) is responsible for selecting origins of DNA replication. In metazoans, origin selection is poorly understood; it is unknown whether specific DNA binding by metazoan ORC controls replication. To address this problem, in vivo and in vitro approaches have been used to demonstrate that Drosophila ORC (DmORC) binds to replication elements that direct repeated initiation of replication to amplify the Drosophila chorion gene loci in the follicle cells of egg chambers. ACE3, a 440-bp chorion element that contains information sufficient to drive amplification, directs DmORC localization in follicle cells. In vivo cross-linking and chromatin immunoprecipitation assays demonstrate association of DmORC with both ACE3 and two other amplification control elements, AER-d and ACE1. To demonstrate that the in vivo localization of DmORC is related to its DNA-binding properties, purified DmORC binds to ACE3 and AER-d in vitro, and like its S. cerevisiae counterpart, this binding is dependent on ATP. These findings suggest that sequence-specific DNA binding by ORC regulates initiation of metazoan DNA replication. Furthermore, adaptation of this experimental approach will allow for the identification of additional metazoan ORC DNA-binding sites and potentially origins of replication (Austin, 1999).

Coordination of replication and transcription along a Drosophila chromosome: Origin recognition complex is localized to specific chromosomal sites, many of which coincide with early activating origins

The mechanisms by which metazoan origins of DNA replication are defined, regulated, and influenced by chromosomal events remain poorly understood. To gain insights into these mechanisms, a systematic approach was developed using a Drosophila high-resolution genomic microarray to determine replication timing, identify replication origins, and map protein-binding sites along a chromosome arm. A high-density genomic microarray was developed that covers the left arm of Drosophila chromosome 2 (representing 20% of Drosophila euchromatic sequence) with 11,243 nearly contiguous 1.5-kb PCR products. Because almost 90% of the nonrepetitive euchromatic sequence from chromosome 2L is represented on this array, it was possible to investigate replication timing at both inter- and intra-genic sequences. A defined temporal pattern of replication was identified that correlates with the density of active transcription. These data indicate that the influence of transcription status on replication timing is exerted over large domains (greater thatn 100 kb) rather than at the level of individual genes. This study identified 62 early activating replication origins across the chromosome by mapping sites of nucleotide incorporation during hydroxyurea arrest. Using genome-wide location analysis, it was demonstrated that the origin recognition complex (ORC) is localized to specific chromosomal sites, many of which coincide with early activating origins. The molecular attributes of ORC-binding sites include increased AT-content and association with a subset of RNA Pol II-binding sites. Based on these findings, it is suggested that the distribution of transcription along the chromosome acts locally to influence origin selection and globally to regulate origin activation (MacAlpine, 2004 ).

The replication timing data revealed clear early and late-replicating domains. These domains were often sharply defined by the density of transcription along the chromosome. The density of RNA Pol II along the chromosome was an order of magnitude greater at the earliest replicating sequences as compared with late-replicating regions. These differences suggest that the molecular architecture of the chromosome may define both the transcription and replication profiles of the chromosome. These transcriptionally active and early replicating domains may be physically marked by a change in chromatin structure that allows for increased access to both replication and transcription factors. It is possible that these domains are defined or restricted by elements of higher order chromosome structure, such as matrix attachment regions, transcriptional insulators, or chromatin loops. However, the state of the chromatin, whether euchromatic or heterochromatic, cannot be the sole determinant for origin activation, since there are examples of efficient heterochromatic origins in S. pombe. Interestingly, the gene-sparse, late-replicating regions identified in Kc cells overlap with late and under-replicated regions found in polytene salivary chromosomes. Taken together, these data suggest that the temporal program of replication is defined by chromatin structure and conserved in different Drosophila cell types (MacAlpine, 2004).

Hydroxylurea (HU) was used to arrest cells early in S phase and to restrict BrdU incorporation to sites overlapping and immediately adjacent to early origins of replication. Using this approach, this study identified 62 sites along the chromosome arm that are used as early replication origins. A recent study observed a change in the local pattern of origin usage at the adenylate deaminase2 locus in response to HU treatment. Although use of HU could have affected the set of origins identified, it is thought that this is unlikely: (1) the pattern of early and late-replicating regions observed using an HU-based protocol is similar to the pattern seen by others using approaches that did not involve replication inhibitors; (2) these studies used arresting concentrations of HU, unlike the hamster cell studies, in which lower concentrations of HU that slowed but did not completely arrest replication were used. Given that only a limited number of sites of BrdU incorporation are found under these arresting conditions, it is likely that only the earliest replicating origins are able to initiate before the intra-S-phase checkpoint prevents other origins from initiating. Finally, even if these origins represent only a subset of the origins along the chromosome, they are a valuable new resource, given the paucity of metazoan origins available prior to these studies (MacAlpine, 2004).

The findings provide clear evidence that ORC is localized to specific chromosomal regions. Consistent with the role of ORC as an essential initiator, this complex is found localized to the majority of early replicating origins, most often at or near the apex of BrdU incorporation. Although ORC was not detected at 27% of the early origins, it is not believed that these represent sites of ORC independent initiation, but rather a limitation of the ChIP technology. By no means have all of the ORC-binding sites along the chromosome been exhaustively identified, since many ORC sites are likely to be occluded from antibody access by additional chromatin-binding complexes. In addition, many ORC-binding sites are likely present in the repetitive and low-complexity sequences that are necessarily omitted from the array (MacAlpine, 2004).

In contrast to S. cerevisiae, where ORC binds discretely to single sites along the chromosome, significant clustering of ORC is seen along the chromosome. Almost 20% of the identified ORC-associated sequences were immediately adjacent to other ORC-associated sequences. This clustering of ORC along the chromosome was also observed at extra chromosomal copies of the ACE3 locus in amplifying follicle cells. Because the ORC-associated sequences often span greater than 3 kb, trivial factors cannot be ruled out such as shear size of the chromatin immunoprecipitated DNA. These clusters of ORC-associated sequence may represent unique chromatin environments favorable to ORC binding, or polymerization of ORC on the DNA following a nucleation event at a specific site (MacAlpine, 2004).

The type of ORC association may influence the nature of replication initiation at a particular locus. It was found that 36% of the early origins contain clusters of three or more ORC-binding sites. For example, at oriDalpha, ORC was continuously associated with a 10-kb region that overlapped a broad region of BrdU incorporation. Interestingly, the analysis of replication intermediates by two-dimensional gel electrophoresis in this region revealed multiple initiation sites over the entire region. In contrast, at the origin identified upstream of the chorion locus, three separate peaks of BrdU incorporation were each marked by distinct ORC-binding sites. This form of ORC association with origins may be analogous to the human lamin B2 locus, where replication initiates at a discrete site. Thus, the origin at oriD and the origin upstream of the chorion locus may represent two distinct types of origins, those defined by broad domains of ORC binding and those associated with more discrete ORC-binding sites (MacAlpine, 2004).

Despite finding ORC at specific regions along the chromosome, the exact mechanism that leads to ORC localization remains to be determined. There are multiple molecular characteristics of the sites of ORC localization, including increased AT-content, noncoding DNA, and RNA Pol II association. These molecular predictors of ORC association could be directly involved in ORC DNA binding, could bind to one or more factors that facilitate ORC localization, or could be required for another origin-related function (e.g., DNA unwinding). It is important to note, however, that none of these attributes are individually sufficient to identify ORC-binding sites. For example, high AT-content by itself is insufficient to define an ORC-binding site, since many sequences on the array have AT-content greater than 62%, but are not represented in the ORC-associated sequences. However, ORC was seemingly excluded from sequences with low AT-content, suggesting that increased AT-content is necessary, but not sufficient for ORC association. Indeed, it has not been possilbe to identify a consensus sequence within the 491 ORC-bound DNA sequences. The lack of a consensus sequence is consistent with the observation that metazoan ORC has only limited sequence specificity in vitro. It is proposed that the attributes that have been identified cooperate to define sites of ORC localization. It is certain that there are additional determinants that were not identified in these studies. For example, the topology of DNA can strongly influence Drosophila ORC binding. Nevertheless, the availability of numerous known Drosophila ORC-binding sites associated with origins of replication will greatly facilitate future studies of ORC localization and origin function (MacAlpine, 2004).

Because only a small subset of origins are likely to initiate in the presence of HU, it is not surprising that only a subset of ORC-binding sites are associated with the early replicating regions. It is anticipated that many of the remaining ORC-binding sites are associated with origins that fire later in S phase. The methods used in this study did not allow the confident identification of late or inefficient origins. However, studies in S. cerevisiae have shown that abrogation of the intra-S-phase checkpoint results in the activation of late-replication origins in the presence of HU, suggesting that a similar approach could be useful for identifying late-activating metazoan origins. In addition, it is possible that a subset of the ORC-binding sites that were identified are involved in other functions, such as gene regulation or the establishment of heterochromatin (MacAlpine, 2004).

It is proposed that the frequent colocalization of ORC and RNA Pol II reflects a connection between transcription and ORC localization. Although it is possible that there is a direct interaction between ORC and RNA Pol II, no such interaction was observed in coimmunoprecipitation assays. In addition, the majority of the sites of RNA Pol II association do not interact with ORC. An alternative hypothesis is that ORC localization is, at least in part, facilitated by a subset of the transcription factors that serve to localize RNA Pol II. Indeed, previous studies have shown that both Drosophila E2F1 and Myb interact with ORC; however, ORC is still localized to the chorion locus during amplification in Myb mutants and mutants of E2F1 that do not interact with ORC. One possible explanation for these findings is that Myb and E2F1 act redundantly to recruit ORC throughout the genome. It is proposed that ORC, like RNA Pol II, can be recruited by many different transcription factors, which would lead to the frequent colocalization with RNA Pol II, but not any particular transcription factor. These factors could recruit ORC by direct interaction or by establishing a chromatin domain that is conducive to ORC recruitment (MacAlpine, 2004).

These findings support a connection between the molecular architecture of the chromosome and the replication process at two levels: (1) the frequent colocalization of ORC and RNA Pol II leads to the hypothesis that nearby transcription factor-binding sites influence the earliest steps of origin selection by facilitating ORC localization and subsequently pre-RC formation; (2) the decision of when each origin initiates replication during S phase (which is mechanistically separate from ORC localization and the assembly of pre-RCs in G1 phase) is connected to transcriptional status in a more global manner. The more transcriptionally active the chromosomal region, the greater the likelihood that replication initiation will occur early in S phase within that domain. The findings indicate that transcriptional status is integrated over broad regions (greater than 100 kb) of the chromosome (rather than individual genes) to determine the time of replication of each chromosomal locus. Further exploration of the connection between higher order chromosome structure and DNA replication will provide insights into the coordination of the molecular events that must occur to propagate and maintain genomic information (MacAlpine, 2004).

Drosophila ORC localizes to open chromatin and marks sites of cohesin complex loading

The origin recognition complex (ORC) is an essential DNA replication initiation factor conserved in all eukaryotes. In Saccharomyces cerevisiae, ORC binds to specific DNA elements; however, in higher eukaryotes, ORC exhibits little sequence specificity in vitro or in vivo. This study investigated the genome-wide distribution of ORC in Drosophila, and ORC was found to localize to specific chromosomal locations in the absence of any discernible simple motif. Although no clear sequence motif emerged, use machine learning approaches could be used to accurately discriminate between ORC-associated sequences and ORC-free sequences based solely on primary sequence. The complex sequence features that define ORC binding sites are highly correlated with nucleosome positioning signals and likely represent a preferred nucleosomal landscape for ORC association. Open chromatin appears to be the underlying feature that is deterministic for ORC binding. ORC-associated sequences are enriched for the histone variant, H3.3, often at transcription start sites, and depleted for bulk nucleosomes. The density of ORC binding along the chromosome is reflected in the time at which a sequence replicates, with early replicating sequences having a high density of ORC binding. Finally, a high concordance was found between sites of ORC binding and cohesin loading, suggesting that, in addition to DNA replication, ORC may be required for the loading of cohesin on DNA in Drosophila (MacAlpine, 2010).

The median distance between ORC binding sites is 11 kb. Changes in the local density of ORC binding dictate whether a sequence will be replicated early or late in S phase, with regions of sparse ORC density replicating later in S phase. The integration of ORC data with the genome-wide mapping of cohesin complex subunits (Misulovin, 2008) revealed extensive colocalization between ORC and multiple cohesin subunits, suggesting that, in Drosophila, ORC may participate in the loading of cohesin on the DNA (MacAlpine, 2010).

ORC binds to specific chromosomal locations in all eukaryotes; however, sequence specificity for ORC has only been observed in the budding yeast, S. cerevisiae. This study identified approximately 5000 ORC-associated sequences distributed throughout the Drosophila genome. Despite being able to accurately resolve the peaks of ORC enrichment to within 1 kb, it was not possible to identify a simple sequence motif like that found for many transcription factors. Instead, a complex code of short sequence k-mers (~6 bases) was sufficient to discriminate between ORC-associated and ORC-free sequences. These short oligomeric sequences likely contribute to a local environment that is favorable for ORC binding. Many of the short sequences that contributed to ORC binding are exclusionary to nucleosomes, suggesting that local nucleosome density may contribute to ORC association. Although open chromatin appears to be important for ORC localization, it is clearly not sufficient for ORC localization as not all predicted sites of low nucleosome occupancy are associated with ORC. Undoubtedly, ORC itself likely contributes some specificity as it has recently been shown that Drosophila ORC6 has a preference for poly(dA) sequences, which are indeed enriched in the top 500 support vector machine (SVM) sequence features (MacAlpine, 2010).

Biochemical studies from multiple metazoan systems have clearly demonstrated that ORC exhibits little sequence specificity in vitro despite a high affinity for DNA. This suggests that a primary determinant of ORC binding will be accessibility to the DNA. A critical difference between the interaction of ORC with DNA in vitro and in the nucleus is that the majority of DNA in the nucleus is packaged into nucleosomes and higher-order chromatin structure. The accessibility of chromatin is an inherited feature established, in part, by primary DNA sequence, transcription factors, insulator elements, transcription machinery, and histone variants. Indeed, this study found that ORC preferentially localizes to open chromatin marked by transcription start sites, the histone variant H3.3, and depletion of bulk nucleosomes (MacAlpine, 2010).

The lack of inherent sequence specificity for ORC may be a unique feature of higher eukaryotes, a feature required to ensure genetic inheritance throughout multiple developmental stages and tissue types. For example, during embryogenesis an increased density of origins is required to ensure the complete duplication of the genome within the confines of a very short cell cycle. By using open chromatin to define potential origins, the cell can harness the plasticity of the transcription program and rapidly reprogram origin selection. Thus, changes in the transcription program will likely affect ORC localization and origin usage. Indeed, numerous examples exist in the literature of origin activity being modulated by transcription (MacAlpine, 2010).

Open chromatin appears to be necessary for ORC binding, but it is clearly not sufficient as many sites of open chromatin are not associated with ORC. Additional proteins may participate in targeting ORC to specific chromosomal locations. Several studies have shown direct interactions between ORC and various transcription factors, including MYB, RBF, and E2F. Although it cannot be ruled out that these and other trans-acting factors are cooperating with open chromatin to localize ORC to the DNA at specific locations, this is not thought to be a general mechanism. For example, no common motif was identified near ORC binding sites. If ORC were being recruited to the DNA by a limited number of transcription factors, evidence would be found for conserved transcription factor motifs at the ORC-associated promoters. The gene ontologies associated with the ORC-associated promoters was explored. No significant functional categories emerged, suggesting that there was not a common regulatory element among the ORC-associated promoters. A consequence of locating the majority of origins at promoter regions is that the replication and transcription forks will be traveling in the same direction. This will eliminate potential head-to-head collisions between DNA and RNA polymerases. This coordination of transcription and replication to avoid direct polymerase collisions is readily apparent in the organization of prokaryotic genomes (MacAlpine, 2010).

The local topology of DNA is a likely factor contributing to the localization of ORC at regions of open chromatin. Negative supercoiling increases ORC's affinity for DNA in vitro by at least an order of magnitude. Negative supercoils are likely to be found behind the promoters of actively transcribed genes where ORC is often localized. Recent genome-wide RNA profiling experiments using sequencing-based platforms have identified the presence of short abortive divergent transcripts at many promoters in mammalian genomes. This divergent transcription would create a very high local concentration of negative supercoils in the vicinity of the promoter. It will be interesting to see if the subset of promoters that are divergently transcribed in mammalian genomes are enriched for ORC and origin activity. Finally, it is tempting to note the parallels between bidirectional DNA replication and bi-directional transcription (MacAlpine, 2010).

The DNA replication program is defined, in part, by where potential origins are established and the time at which they are activated in S phase. Studies in S. cerevisiae, Drosophila, and human cells have clearly shown that the local chromatin environment influences origin activation. For example, tethering the histone deacetylase, RPD3, to Ori-beta at the chorion locus inhibits origin function. This study shows that the density of ORC on the chromosome also contributes to the overall temporal pattern of DNA replication. Not unexpectedly, sequences distant from ORC binding sites replicate later in S phase and are limited to gene-poor regions of the genome. The localization of ORC to promoters of actively transcribed genes results in a higher density of ORC in gene-rich regions, which guarantees their duplication early in S phase. Mammalian replication timing studies have shown that the rate of mutagenesis is significantly higher in late S phase. By duplicating gene-rich regions in early S phase, the cell can specifically maintain the integrity of these regions of the genome (MacAlpine, 2010).

These genome-wide studies of ORC binding in Drosophila revealed extensive colocalization between ORC and multiple subunits of the cohesin complex. ORC was most often found at the center of cohesin peaks, suggesting that ORC may participate directly or indirectly in sister-chromatid cohesion. Prior genome-wide mapping experiments in mammalian cells implicated the insulator element, CTCF, in sister-chromatid cohesion. In contrast, in Drosophila, <15% of the CTCF sites are coincident with the cohesin complex member, SA; however, there is significant overlap between the CP190 insulator element and cohesin binding sites. In egg extracts from Xenopus, ORC and pre-RC assembly are required for cohesin loading on sperm chromatin. Despite inhibiting pre-RC assembly by depleting double parked by RNAi, it was not possible to perturb the levels of chromatin-associated Nipped-B, SMC1, and SA. These results suggest that in Drosophila the loading of cohesin is not dependent on pre-RC formation. The role of Drosophila ORC in sister-chromatid cohesion could not be directly tested because of compounding cell cycle phenotypes induced by RNAi depletion of ORC subunits. Alternatively, in the absence of pre-RC formation, cohesion may still be established, possibly at different locations, via an alternative pathway. Future experiments will address the localization of cohesin subunits in the absence of pre-RC assembly (MacAlpine, 2010).

Binding of Drosophila ORC proteins to anaphase chromosomes requires cessation of mitotic cyclin-dependent kinase activity

The initial step in the acquisition of replication competence by eukaryotic chromosomes is the binding of the multisubunit origin recognition complex, ORC. A transgenic Drosophila model is described which enables dynamic imaging of a green fluorescent protein (GFP)-tagged Drosophila melanogaster ORC subunit, DmOrc2-GFP. It is functional in genetic complementation, expressed at physiological levels, and participates quantitatively in complex formation. This fusion protein is therefore able to depict both the holocomplex DmOrc1-6 and the core complex DmOrc2-6 formed by the Drosophila initiator proteins. Its localization can be monitored in vivo along the cell cycle and development. DmOrc2-GFP is not detected on metaphase chromosomes but binds rapidly to anaphase chromatin in Drosophila embryos. Expression of either stable cyclin A, B, or B3 prevents this reassociation, suggesting that cessation of mitotic cyclin-dependent kinase activity is essential for binding of the DmOrc proteins to chromosomes (Baldinger, 2009).

DmOrc2 was chosen for two reasons. First, it constitutes an essential part of the ORC core. As such, it was expected to better reflect the localization of the complex in its origin-defining function compared to peripheral ORC subunits. Second, several null alleles of k43, the DmOrc2 gene, have been identified, allowing pursuit of a genetic complementation strategy to verify transgene functionality. Rescue transgenes consisted of genomic copies of the DmOrc2 gene in which GFP was inserted in frame, coding for either an N- or C-terminal fusion of the ORC subunit. Using complementation as the most conclusive genetic criterion, both fusions proved functional. This indicates DmORC's flexibility to accommodate substantial heterologous protein moieties, exemplified in this study by GFP attached in two independent positions within the complex structure. In particular for a protein like DmOrc2, functioning as an integral part of a multiprotein complex, it was essential to avoid an overexpression situation, as in the absence of authentic binding partners its fluorescence signal is expected to mislocalize. Thus, fly lines with physiological DmOrc2 expression levels and the quantitative participation of the fusion protein in the holo- or the core complex were a prerequisite to address the cell cycle events governing DmORC's interaction with chromosomes by a biologically meaningful experimental approach. Both criteria were met in the DmOrc2-GFP transgenic as well as the rescue lines. Thus, for all parameters tested, the Drosophila model reflected faithfully the behavior of endogenous DmOrc2, allowing the visual tracing of DmORC-GFP. The use of the term 'DmORC-GFP' therefore refers to both the DmORC core and the holocomplex formed upon DmOrc1 association, which cannot be distinguished by the chosen imaging approach (Baldinger, 2009).

Aside from ensuring the congruence between transgenic and endogenous ORC subunits, the experimental strategy of tracking ORC during the cell cycle also avoids ambiguities sometimes associated with the fixation or physiological stressing of cells and organisms. Notwithstanding such methodological issues, the observed dynamics of ORC-chromatin interactions reported previously suggested a significant divergence between different biological systems. In yeast, ORC binds chromatin throughout the cell cycle, including metaphase, as has also been reported for embryonic Drosophila and mammalian ORC core subunits. Other studies came to the conclusion that members of the mammalian core complex are mostly excluded from metaphase chromatin, similar to Xenopus and also Drosophila larval neuroblasts. Based on immunolocalization studies, the latter analysis came also to the conclusion that DmOrc2 accumulates on late anaphase/telophase chromosomes, similar to the current findings. Differences in the reported localization patterns of ORC can possibly be explained by cell-type-dependent or, in particular, by interspecies variations in the control mechanism of the cell division cycl (Baldinger, 2009).

From the imaging analysis in this in vivo model, it is concluded that the majority of DmORC-GFP is displaced from the chromosomes in early mitosis and diffusely distributed throughout the cell without any recognizable localization pattern. Therefore, current models of the embryonic Drosophila ORC cycle should be scrutinized when they place the core DmORC on mitotic chromosomes. Toward the end of mitosis, DmORC-GFP is chromatin bound again, and this relocalization seems to be quantitative within the detection limits of the methodology employed. No principal differences were observed in this dynamic behavior of DmORC-GFP between syncytial and cellularized stages of embryonal development (Baldinger, 2009).

Proteolytic control of ORC core subunits has not been reported so far. In line with this lack of evidence, this study does not indicate that DmORC-GFP levels are subject to mitosis-specific protein degradation (as are other regulators of cell cycle progression), with the fluorescence signal of DmORC-GFP clearly visible in early mitosis, before gradually refocusing on late mitotic chromosomes. This entire process might be completely attributed to control over intracellular localization of DmORC-GFP during the cell cycle. However, while a substantial resynthesis of DmORC core subunits appears unlikely given the observed timing of this process, in particular with the additional requirements for complex assembly and chromophore maturation, a partial destruction of core DmORC subunits, followed by chromosomal recruitment of DmORC from cytoplasmic pools at the onset of a new round of pre-RC formation, cannot be ruled out (Baldinger, 2009).

Origin specification and pre-RC assembly in eukaryotes start with the chromatin binding of ORC. This study showed the cell-cycle-dependent changes of DmORC-GFP localization in embryos. Its rapid accumulation on chromosomes is detectable by late anaphase when CDK activity drops to the low levels observed in the late M and early G1 phases. The dependence of DmORC-GFP chromosome binding on low CDK activity was established by following the fluorescence signal upon cell cycle arrest in response to the expression of stable mitotic cyclins A, B, and B3, which are not subject to proteasomal degradation. Their presence prevented chromatin binding of DmORC-GFP. Previous reports describing the reloading of ORC to late mitotic chromatin in various cellular systems of metazoan origin have implicated mitotic CDKs in this process, supported by corresponding biochemical analyses. In Drosophila, it is known that the expression of individual stable cyclins does not interfere with the cell-cycle-controlled degradation of the endogenous cyclins. Thus, this in vivo analysis allows extension of the general assumption of a role for mitotic CDK involvement in triggering the start of pre-RC assembly to specifically conclude that all mitotic CDK/cyclin activities have to cease for DmORC-GFP to become chromatin bound (Baldinger, 2009).

How can this dynamic behavior of DmOrc2 be interpreted in the light of previous observations regarding the APC-dependent degradation of DmOrc1 in late mitosis, only to reemerge in late G1? Even when considering that metazoan Orc1 often shows expression, localization, and turnover patterns independent of other ORC subunits, reflecting temporal events in the control over ORC activity, the almost converse mitotic shuttling patterns of DmORC subunits are somewhat surprising. It should be noted, however, that DmOrc1-GFP could also be detected on telophase chromosomes before being degraded. Most studies of metazoan ORC concur that Orc1 is essential to establish initial DNA binding of ORC and subsequent steps of pre-RC formation, supported by the recent finding that elevated Orc1 levels can actually promote binding of endogenous Orc proteins during late mitosis. It is conceivable that in Drosophila this process takes place during a brief time window in late mitosis and could be sufficient to trigger the recruitment of other pre-RC proteins, which according to most analyses occurs prior to late G1. Alternatively, the remaining chromatin-bound DmORC core might be sufficient to promote completion of the pre-RC. From these lines of reasoning, it is already obvious that further experiments, in directly comparable settings for both the experimental protocols followed as well as for the cell types and developmental stages analyzed, will be required to resolve this issue. This will be facilitated by the availability of Drosophila orc1-/- lines (Baldinger, 2009).

After this initial step in pre-RC assembly, other replication initiation proteins have to be loaded on chromosomes for them to become licensed for replication. Among these factors is the heteromultimeric minichromosome maintenance (MCM) complex, associated with a DNA helicase activity. Previous immunolocalization studies of the association/dissociation cycles of Drosophila MCM demonstrated their binding to mitotic chromatin upon cell cycle arrest by expression of stable cyclin B, corresponding to early anaphase stages. Assuming an unconditional requirement for prebound ORC for MCM chromatin binding, the current data would predict MCM binding at later cell cycle stages, after cessation of mitotic CDK activity. At first glance, these results on the timing of MCM-chromatin association might not be easy to reconcile with the current findings but can be explained by (1) the influence of the imaging methodology as describe for DmORC localization, (2) different sensitivity thresholds of the detection systems, or (3) potential uncharacterized effects of the stable cyclin-CDK complexes used in different studies. In any case, no real discrepancy is seen, since chromatin loading of MCM proteins in unperturbed cell cycles has only been evident in late anaphase/telophase, fully compatible with the current results for DmORC in both perturbed (i.e., stalled by stable cyclin expression) and unperturbed cell cycles (Baldinger, 2009).

It will be interesting to determine if this binding is partly responsive to potential changes in chromosome structure occurring as mitotic chromosomes pass toward telophase or whether DmORC responds directly or indirectly to changes in the kinase environment of late mitotic cells. The latter possibility would argue that a decrease in DmORC's phosphorylation state results in its increased affinity to chromatin. This scenario appears attractive since it has been demonstrated that in vitro binding of DmORC to DNA is strongly diminished whenever it is phosphorylated by various CDK/cyclin activities. Combined with the current cytological studies, these findings make mitotic CDKs attractive candidate kinases for actively suppressing DmORC binding to chromatin. This view is also in line with the localized cyclin destruction in syncytial cell cycles of Drosophila. The resulting abrogation of CDK1 activity in the vicinity of the mitotic spindle can be monitored by the distribution of phospho-histones, akin to the observed gradual rebinding of DmORC, starting from the centromeric regions of anaphase chromosomes (Baldinger, 2009).

In summary, this study report the spatial and temporal dynamics of the initiator protein ORC in a live metazoan organism. Along with the cell cycle, ORC periodically associates with and dissociates from chromatin. The initial interaction in preparation for the next chromosome cycle occurs in late anaphase. This binding of ORC to chromatin depends critically on the cessation of mitotic cyclin activity, linking this first step of replication licensing to the CDK-driven control pathways of cell cycle progression. Finally, it is obvious that different mechanisms evolved between species controlling the activities of ORC. While all of them are compatible with the general requirements for origin definition, pre-RC assembly, and the prevention of rereplication, it cautions against the extrapolation of findings from one experimental system to another. This underscores the value of multipurpose in vivo models like the one described in this study, allowing a comprehensive approach for probing ORC functions. Its use should not be restricted to further exploring ORC in DNA replication initiation, but it should also be useful to study ORC's role in proliferation and in the development of an organism (Baldinger, 2009).

Chromatin signatures of the Drosophila replication program

DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, relatively little is known about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. This study describes the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. While the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. Chromatin features were identified that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. Accurate and predictive models were generated from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events (Eaton, 2011).

The chromatin landscape clearly impacts both the expression and the replication of the genome. For example, the transcriptionally active euchromatin typically replicates prior to the repressed heterochromatic sequences. Studies in yeast, Drosophila, and mammalian systems have shown that changes in histone acetylation are associated with changes in the replication program. However, a comprehensive view of the replication program in the context of chromatin modifications and DNA-binding proteins is lacking (Eaton, 2011).

The different modENCODE data types across multiple cell lines (The modENCODE Consortium 2010) allowed the definition of the chromatin and transcription landscape associated with features of the DNA replication program. For each replication data type (replication timing, early origins, and ORC binding), a 43 × 3 matrix was generated, with each column representing a specific cell line and each row representing the enrichment or correlations with chromatin marks, DNA-binding proteins, nucleosome density, histone variants, nucleosome turnover (CATCH-IT), and gene expression (RNA-seq). For the replication timing profiles where there are no discrete peak calls, the Spearman's correlation was calculated between each factor with the whole-genome replication timing profile. For early origins of replication and ORC-binding sites, the median log2 enrichment was calculated of each factor within all BrdU peaks and within 500 bp of ORC ChIP-seq peak centers, respectively (Eaton, 2011).

The selection and regulation of DNA replication origins was found to be associated with distinct sets of chromatin marks and DNA-binding proteins. Prior studies have associated early replication with active transcription and the presence of 'activating' chromatin modifications such as histone acetylation, whereas late replication is associated with 'repressive' chromatin marks such as those found in the heterochromatin. Indeed, this study found that gene expression is positively correlated with replication timing, as are generally euchromatic marks such as H3K4me1 and H3K18ac. In contrast, heterochromatic marks such as H3K27me3 and H3K9me2 are negatively correlated with replication timing. The sequences surrounding early origins were also enriched for activating chromatin marks as well as specific DNA-binding proteins, including chromatin remodeling factors (Eaton, 2011).

Because many of the ORC-binding sites colocalized with promoters of active genes, the ORC-binding sites were separated into those that are TSS proximal (within 1 kb of a TSS) and those that were not at a TSS (distal). Particular interest was placed on chromatin features that are shared between ORC-binding sites both proximal and distal to promoters. Additionally, marks that are specific to ORC sites distal from a promoter will be of interest, as these marks may be required for ORC binding or function in the absence of a promoter (Eaton, 2011).

ORC-binding sites proximal to TSSs were enriched for chromatin remodelers such as the NURF complex (NURF301 [also known as E(BX)], ISWI) as well as other DNA-binding proteins such as GAF, RNA Pol II, and CHRO. These TSS-associated ORC sites were also enriched for H3K9ac, H3K27ac, H3K4me2, and H3K4me3 -- marks frequently found at promoters. Interestingly, those ORC sites that did not overlap with a TSS (distal) were also enriched for chromatin remodelers ISWI and NURF301, as well as GAF, which has also been implicated in chromatin remodeling. Consistent with the idea of ORC localizing to dynamic and active chromatin, an enrichment was found for CATCH-IT and H3.3 at ORC sites both proximal and distal to TSSs, as well as a reduction in bulk nucleosome occupancy. ORC sites not located at promoters were enriched for many of the same histone marks as those at promoters, with a few notable exceptions. A decrease in H3K4me3 was found at ORC sites distal from a promoter, as well as an increase in H3K18ac and H3K4me1 (Eaton, 2011).

Chromatin features specific to transcription start sites such as RNA Pol II and H2Av were decreased at ORC-binding sites distal to promoter elements. A small amount of RNA Pol II signal remained in the TSS distal ORC-binding sites; however, in comparison to the local enrichment of ISWI and GAF, there was a clear reduction in local signal. The remaining signal may be due to unannotated transcription start sites (Eaton, 2011).

Chromatin marks that are associated with active transcription through gene bodies (e.g., H3K79me1, H3K36me1, and H3K36me3) were not found above background levels at any ORC-binding sites. However, H3K36me1 was found specifically flanking those ORC-binding sites that did not coincide with a TSS. ORC has been shown to facilitate the formation of heterochromatin and HP1 binding; however, ORC sites were depleted for heterochromatic histone modifications such as H3K27me3 and H3K9me2/3 and were only slightly enriched for HP1. This may be due, in part, to the inability to map distinct ORC-binding sites in repetitive sequences, a current limitation of high-throughput sequencing approaches (Eaton, 2011).

The chromatin signatures were examined of promoter elements with and without ORC associated to determine whether there were unique chromatin signatures specific for ORC associated promoters. Since those promoters with proximal ORC binding tend to be far more actively transcribed than those without ORC, the comparison was limited to active promoter elements only. It was found that ORC-associated promoters had modestly increased chromatin remodeling activities, decreased nucleosome occupancy, and greater evidence of nucleosome turn-over relative to other active promoters not associated with ORC. In summary, these results indicate that dynamic chromatin environments may contribute to ORC localization and the subsequent activation of replication origins (Eaton, 2011).

Integrative analysis of gene amplification in Drosophila follicle cells: parameters of origin activation and repression

In metazoans, how replication origins are specified and subsequently activated is not well understood. Drosophila amplicons in follicle cells (DAFCs) are genomic regions that undergo rereplication to increase DNA copy number. All DAFCs were identified by comparative genomic hybridization, uncovering two new amplicons in addition to four known previously. The complete identification of all DAFCs enabled investigation of these in vivo replicons with respect to parameters of transcription, localization of the origin recognition complex (ORC), and histone acetylation, yielding important insights into gene amplification as a metazoan replication model. Significantly, ORC is bound across domains spanning 10 or more kilobases at the DAFC rather than at a specific site. Additionally, ORC is bound at many regions that do not undergo amplification, and, in contrast to cell culture, these regions do not correlate with high gene expression. As a developmental strategy, gene amplification is not the predominant means of achieving high expression levels, even in cells capable of amplification. Intriguingly, it was found that, in some strains, a new amplicon, DAFC-22B, does not amplify, a consequence of distant repression of ORC binding and origin activation. This repression is alleviated when a fragment containing the origin is placed in different genomic contexts (Kim, 2011; full text of article).

CycA is involved in the control of endoreplication dynamics in the Drosophila bristle lineage

Endocycles, which are characterised by repeated rounds of DNA replication without intervening mitosis, are involved in developmental processes associated with an increase in metabolic cell activity and are part of terminal differentiation. Endocycles are currently viewed as a restriction of the canonical cell cycle. As such, mitotic cyclins have been omitted from the endocycle mechanism and their role in this process has not been specifically analysed. In order to study such a role, this study focused on CycA, which has been described to function exclusively during mitosis in Drosophila. Using developing mechanosensory organs as model system and PCNA::GFP to follow endocycle dynamics, it was show that (1) CycA proteins accumulate during the last period of endoreplication, (2) both CycA loss and gain of function induce changes in endoreplication dynamics and reduce the number of endocycles, and (3) heterochromatin localisation of ORC2, a member of the Pre-RC complex, depends on CycA. These results show for the first time that CycA is involved in endocycle dynamics in Drosophila. As such, CycA controls the final ploidy that cells reached during terminal differentiation. Furthermore, the data suggest that the control of endocycles by CycA involves the subnuclear relocalisation of pre-RC complex members. This work therefore sheds new light on the mechanism underlying endocycles, implicating a process that involves remodelling of the entire cell cycle network rather than simply a restriction of the canonical cell cycle (Sallé, 2012).

The data reveal a role for CycA in the control of S phase in Drosophila. Until now, CycA has been considered exclusively as a mitotic cyclin in this organism. Both up- and downregulation of CycA levels reduces the number of endocycles. In particular, the transition between early and late S phase is either advanced or delayed after CycA up- or downregulation respectively. In addition, the duration of the late S phase is increased under both conditions. The combination of these effects leads to a reduction in ploidy. Finally, it was shown that CycA controls the localisation of the pre-replicative factor ORC2, an observation that may explain the effects of CycA on endocycle dynamics. All of these observations were performed under CycA hypomorphic conditions that do not affect mitoses. Collectively, these results show that the mechanism underlying endocycles requires a very low level expression (even undetectable) of major cell cycle regulators such as CycE and CycA (Sallé, 2012).

In Drosophila, CycA has been found in exclusive association with Cdk1, rather than with both Cdk1 and Cdk2 as in mammals. As such, CycA has been always considered as a mitotic cyclin. Several observations suggest that CycA is implicated in DNA replication, however. CycA overexpression can trigger S phase, even in a CycE mutant background. This study shows that CycA has a bona fide function that influences the progression of the S phase, and that more precisely involves the control of late S-phase timing during endocycles. Two possible mechanisms could account for these results: either CycA affects components required for overall S-phase progression or it modulates the activity of specific members of the core replication machinery. The former hypothesis is supported by data showing that in Drosophila Cdk1 and CycA cooperatively inhibit transcriptional activation by affecting the essential S-phase regulator E2F1. In addition, degradation of E2F1 is related to its CycA-dependent phosphorylation in mammalian cells. However, during endocycles in the bristle lineage, neither overexpression nor downregulation of CycA had a significant effect on the level of E2F1 accumulation. Hence, the results favour a mechanism where CycA regulates specific components of the replication machinery. Indeed, CycA was shown to be required for the relocalisation of the pre-RC protein ORC2 during the early-to-late S-phase transition (Sallé, 2012).

Classically, ORC2 is considered to be part of the pre-RC. However, the pre-RC component MCM5 and the replication factor PCNA::GFP do not undergo CycA-dependant control of their localisation, as was observed for ORC2. Although the possibility that localisation of Cdc6 or Cdt1 (or other components) might depend on CycA activity cannot be ruled out, these data suggest that ORC2 acts independently of the pre-RC. Indeed, apart from its well-known role in DNA replication, ORC2 has been shown to participate in heterochromatin maintenance. Moreover, ORC proteins localise to heterochromatin in vivo and interact directly with the essential heterochromatin factor HP1. As such, ORC2 could act as a gatekeeper between euchromatin and heterochromatin or, alternatively, it could be implicated in the establishment or the maintenance of chromatin structure. After CycA overexpression, the observed lengthening of late S-phase is probably due to stable relocalisation of ORC2 to the heterochromatin that, concomitant with the high CycA activity, induces a complete replication of heterochromatin. In addition, under conditions of CycA loss of function where ORC2 does not relocalise to heterochromatin, it was often observed that chromosomes were under-condensed. Similarly, irregularly condensed mitotic chromosomes were observed in Drosophila and in mammalian ORC2-depleted cells. In any case, the fact that heterochromatin can still be formed in the absence of ORC2 relocalisation suggests that ORC2 is necessary but not essential for heterochromatin maintenance. This dispensability of ORC2 has been also observed for endoreplication, as, in orc2 mutants, endocycles occurred but cell ploidy was reduced. These data are therefore in agreement with results showing that CycA controls ORC2 localisation and modulates endocycles dynamics. Thus, lengthening of the late S-phase observed after CycA mis-regulations probably reflects a dual function for ORC2 in both DNA replication and chromatin structure (Sallé, 2012).

These data show that the timing of the early S phase was also affected under conditions of CycA mis-regulations. Early S-phase was lengthened under the CycA downregulation condition and conversely shortened after CycA overexpression. As the data showed that CycA begins to accumulate at the end of early S phase, this suggests that CycA controls the transition between early and late S phase. Under CycA downregulation conditions, it was observed that ORC2 remained throughout the nucleus. This maintenance of ORC2 in the euchromatic regions may explain the delay in transitioning from early to late S phase as due to the stabilisation of a permissive state for euchromatin replication. Strengthening the case that CycA is important for this process, DSBs detected by gammaH2AV immunoreactivity are also induced under these conditions. These ectopic DSBs were not associated with ectopic heterochromatin foci, arguing against them being caused by heterochromatin boundaries. This suggests that DSBs reflect partial replication due to the low level of Cdk activity. As such, the results imply that CycA is involved in the control of DNA replication per se. These results are in agreement with recent data of Ding and MacAlpin (2010) on DNA re-replication, suggesting that Cdk/CycA activity specifically inhibits pre-RC assembly in the euchromatin (Sallé, 2012).

Using DSBs to assay for replication defects, it was shown that ORC2 overexpression also induced similar replication impairment as CycA depletion. These results suggest that CycA-dependent ORC2 intra-nuclear re-localisation would be a way to deplete euchromatin of ORC2, concluding replication and generating a period during which euchromatic DNA is unable to relicense. This model is also consistent with observations made in other systems, for example, in CHO hamster cells where Cdk1/CycA was able to hyper-phosphorylate ORC1 and decrease its affinity for chromatin. Moreover, it has also been shown that Cdk1/CyclinA can phosphorylate ORC2 in vitro. All these data support a model where CycA, in a dose-dependent manner, promotes ORC2 shuttling between euchromatin and heterochromatin as a mechanism to accomplish the early S phase (Sallé, 2012).

These data complete the current model of endocycle progression by incorporating the mitotic factor CycA. In this model, CycE activity oscillations, mediated in part by E2F1 activity, drive cyclic anti-parallel oscillations of APC/C-Fzr activity. Periods of low APC/C activity would permit accumulation of pre-RC complex proteins, such as ORC1 and Cdc6. As such, these oscillating activities generate S- and G-phase time windows, which ensure that the genome is properly replicated once in every cycle. It is suggest that CycA reinforces the exit from the replicative state, probably by controlling localisation and/or activity of pre-RC factors such as ORC2 (Sallé, 2012).

Finally, this work reveals a novel vision for the mechanism underlying endocycles. This mechanism appears not to be a subset of that controlling mitosis. Rather, this study show that, as the same factors participate in both processes, the mechanism involved in endocycles and mitotic cycles differs only by quantitative variations in protein levels and probably by the temporal expression of these factors (Sallé, 2012).

Protein Interactions

About half of the ORC separates into a high-molecular-weight fraction estimated to be greater than 500 kD, whereas the remainder appears in complexes of lower molecular weight. ORC2 is consistently associated with 5 other proteins. The sum of their molecular weights is a predicted 395 kD, compared to the 413 kD estimated for the yeast ORC complex. A second member of the fly ORC complex, has been cloned based on sequence homology to ORC5 of yeast. The fly protein contains a purine nucleotide binding site P-loop (Gossen, 1995).

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions: only rudimentary molecular details of its structure are available. A powerful tool in the analysis of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent on other proteins. To examine HP1 interacting proteins, three distinct oligomeric species of HP1 have been isolated from the cytoplasm of early Drosophila embryos and their compositions analyzed. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae (Huang, 1998).

To isolate the genes encoding the unknown subunits of Drosophila origin recognition complex (ORC), ORC was purified from Drosophila embryos (0-12 hr of development) through the several steps of conventional chromatography. Proteins corresponding to Drosophila DmORC3 (79 kD), DmORC4 (42 kD), and DmORC6 (30 kD) were isolated and tryptic peptides sequenced. On the basis of sequence information, degenerate primers were designed and used to amplify the genomic DNA that encodes the peptide. These DNAs were used to probe a Drosophila melanogaster cDNA library. Two different peptides from each subunit band were used to make such genomic probes. For each set, a single ORF was identified that encodes all of the peptides derived from the appropriate subunit of ORC. An intact cDNA for each subunit included a putative initiator ATG preceded by stop codons in all three reading frames. In combination with previously described DmORC1, DmORC2, and DmORC5 genes, the isolation of the Drosophila cDNAs for ORC3, ORC4, and ORC6 completes the identification of the genes encoding for this complex (Chesnokov, 1999).

Translation of full-length cDNA clones for each subunit predicts a range of amino acid identities with yeast ORC components (24% for ORC4, 21% for ORC3, and 19% for ORC6). The alignments of amino acid identities between Drosophila ORC3 and ORC6 components with counterparts in the budding yeast complex are not compelling and one must hold open the possibility that selection might have substantially allowed for divergence of certain functions. Alternatively, the subunits for ORC3 and ORC6 described for Drosophila might not be orthologs with the S. cerevisiae genes at all and may have derived from another evolutionary branch. This is more likely to be the case for ORC6, where the yeast and Drosophila proteins have very different sizes and show no patches of statistically significant identity or homology. It will be interesting to learn if a gene similar to Drosophila ORC6 is found in other organisms. The ORC3 alignments do show that both humans and Xenopus have orthologs to the Drosophila ORC3. The Xenopus p81 protein was initially identified as an ORC2-associated protein, and recent work confirms that this protein is part of a larger ORC complex in Xenopus (Chesnokov, 1999).

The metazoan ORC4 genes show several regions of peptide identity to one another and to the homologous protein in yeast. In particular the ORC4 proteins of human and Drosophila, and a recently characterized Xenopus homolog conserve ATP-binding and hydrolysis motifs constituting the Walker A and B boxes and other extensive homologies in this central domain. The S. cerevisiae ORC4 protein maintains good homology around the Walker B motif, whereas more divergence is apparent at the A box. It seems possible that an unknown ORC4 ATP binding function was shared in a common ancestor and perhaps preserved in the metazoans. This activity might have been lost in the budding yeast because mutation of these more divergent motifs in S. cerevisiae seems to be of no functional consequence (Chesnokov, 1999 and references).

To confirm that the genes identified are those encoding the proteins copurifying in the complex, polyclonal antisera specific for each of the individual full-length proteins were raised. Purified ORC was subjected to SDS-PAGE analysis together with embryo extracts immunoprecipitated with either ORC2 or ORC6 antibodies. The proteins were prepared for immunoblot analysis using individual anti-ORC subunit antibodies. Each of the reagents specifically recognizes the expected proteins. An interesting point is that in comparison to other ORC subunits there seems to be a free pool of ORC6 unassociated with the other components. Biochemical fractionation indicates that ORC6 is the only subunit maintained in the extracts in a low molecular weight form unassociated with other DmORC components (Chesnokov, 1999).

In principle, the genetic complexity of origin of DNA replication (ori) usage in Drosophila might be explained by the existence of a large family of ORC genes, each with a distinct pattern of temporal or tissue expression and the ability of different ORC complexes to recognize different DNA elements. No data consistent with this thought were obtained. Each of the ORC genes is unique in Drosophila. As anticipated, large stores of mRNA for each of the ORC genes are maternally deposited and the level of such mRNA decreases through development. Undoubtedly, zygotic induction of ORC genes is highly regulated in a tissue-specific manner and the inability to detect mRNA in the latter stages is probably due to the insensitivity of the Northern blotting procedure. A hint of such differential and complex regulation is indicated by an increase in abundance for ORC2 and ORC4 mRNAs at ~6 hr of development, relative to that detected at 4-6 hr. Also, a second transcript for both ORC4 and ORC6 becomes more apparent at these times. The biological significance of these second transcripts requires further study. It is suspected that these second transcripts represent alternate start sites or 3' processing of the mRNAs encompassing the single ORFs, because protein patterns from the immunoprecipitations of the ORC material are identical throughout the early staged times, and attempts to clone cDNAs from other staged libraries yield the same ORFs (Chesnokov, 1999).

With complete cDNAs for each of the Drosophila ORC subunits available it was of interest to see if coexpression of the genes from baculovirus vectors would be sufficient for complex formation. Each of the genes were individually expressed and only ORC2 and ORC6 were found to be readily soluble proteins. However, upon coinfection of all six viral vectors, each of which carry a unique ORC subunit gene, all other proteins (i.e., ORC1, ORC3-ORC5) remain soluble and readily form a complex. A His-tagged version of ORC1 was used to simplify purification. This material was purified further by sedimentation through a glycerol gradient. The six subunits cosediment as does the native material (Chesnokov, 1999).

To date, the best understood biochemical activity of S. cerevisiae ORC is its ATP-dependent sequence-specific DNA binding. Employing an immunoprecipitation method and DNA restriction fragments that span the ACE3 and oribeta elements of the chorion gene cluster of Drosophila no evidence could be obtained for site-specific DNA-binding activity for the recombinant or embryonic DmORC (M. Gossen and M. Botchan, cited in Chesnokov, 1999). These results may be anticipated from the known rapid and permissive replication ori usage in early embryogenesis (Chesnokov, 1999).

A soluble DNA replication system that is dependent on the Drosophila ORC protein would be the most direct assay for the functional integrity of the reconstituted complex. X. laevis soluble egg extracts have provided a powerful tool to study cell-cycle and DNA replication proteins; therefore, attempts were made to mimic such protocols with early embryonic extracts of Drosophila. Xenopus demembraned sperm DNA was used as a template for replication to be mediated by Drosophila 0- to 2-hr embryonic extracts. DNA synthesis in these extracts is at least 5-10 times less efficient than that in the synchronized Xenopus egg extract, in side-by-side reactions. The formation of nuclei around the sperm chromatin in Drosophila extracts is low compared to that observed in Xenopus extracts. Accordingly, a severalfold enhancement of DNA replication is observed when a Xenopus membrane fraction is added to the soluble Drosophila extracts. DNA replication in these extracts is ORC dependent (Chesnokov, 1999).

In Drosophila, four unknown proteins from embryonic extracts copurify (and cosediment) with DmORC2 and DmORC5, suggesting that these form DmORC (Gossen, 1995). One 79 kDa protein component is similar to the molecular weight of Latheo, the Drosophila ORC3. Immunoprecipitation of DmORC2 from Schneider cells results in specific coimmunoprecipitation of Lat. Immunoprecipitation of Lat with anti-Lat antibodies also coimmunoprecipitates DmORC2. The sequence similarity with ScORC3, the association of Lat with DmORC2, and the cell proliferation defects of lat null mutants strongly argue that Lat functions as a subunit of Drosophila ORC (Pinto, 1999).

The E2F transcription factor and retinoblastoma protein control cell-cycle progression and DNA replication during S phase. Mutations in the Drosophila E2f1 and DP genes affect the origin recognition complex (DmORC) and initiation of replication at the chorion gene replication origin. Mutants of Rbf (an retinoblastoma protein homolog) fail to limit DNA replication. DP, E2f1 and Rbf proteins are located in a complex with ORC, and E2f1 and ORC are bound to the chorion origin of replication in vivo. These results indicate that E2f and Rbf function together at replication origins to limit DNA replication through interactions with ORC (Bosco, 2001).

To explore the possibility that E2f-Rbf is directly involved in controlling ORC activity, a test was performed to see whether a female-sterile mutant of Rbf (Rbf120a) has DNA replication and gene amplification defects in follicle cells of the Drosophila ovary. TheRbf120a mutation is due to a P-element insertion that causes reduced levels of wild-type Rbf protein, and Rbf14 is a null mutant. Ovaries from mutant Rbf120a/Rbf14 and heterozygous Rbf14/+ females were double labelled with 5-bromodeoxyuridine (BrdU) and anti-ORC2. Wild-type Drosophila follicle cells undergo endoreduplication cycles (endo cycles), reaching 16n ploidy by stage 9 or 10A of egg-chamber development. In stage 10B follicle cells, endo cycles have ceased, ORC has been cleared from the nucleus, and ORC is localized to discrete genomic regions undergoing amplification. Amplification is detected by BrdU incorporation at ORC localized foci. By contrast, the Rbf120a/Rbf14 mutant egg-chambers have a mosaic of follicle cells exhibiting striking replication defects: (1) some mutant follicle cells have inappropriate total nuclear ORC2 staining and continued endo cycles instead of amplification; (2) some follicle cells with specific ORC2 localization to replication origins have undergone gene amplification; and (3) some cells perform both amplification and genomic replication. Staining ovaries with anti-Rbf antibodies reveals a uniform absence of Rbf protein, and thus the mosaic phenotype cannot be explained by stochastic differences in Rbf protein levels (Bosco, 2001).

Whether E2f-Rbf complexes execute an S-phase function through a direct interaction with ORC was tested. Immunoprecipitations were carried out on ovary extracts; immunoblots of the pellets show that E2f and Rbf co-immunoprecipitate with Drosophila ORC when either anti-ORC2 or anti-ORC1 antibodies were used. The E2f-Rbf-ORC interaction could also be detected when immunoprecipitation reactions were performed with anti-E2f polyclonal or anti-DP monoclonal antibodies. This complex could be specifically immunoprecipitated from ovary extracts with five different antibodies. It is possible that in extracts the dDP-E2f-Rbf and ORC interaction might be due to dDP-E2f and ORC binding next to each other on DNA fragments. Therefore, immunoprecipitation reactions were carried out in the presence of ethidium bromide or micrococcal nuclease to disrupt protein-DNA interactions or cleave DNA fragments. Treatment of immunoprecipitation reactions with either reagent failed to disrupt the E2f-Rbf-ORC interaction. Furthermore, a mutation in DP predicted to reduce the DNA-binding activity of E2f did not abolish the E2f-Rbf-ORC interaction. It is therefore concluded that E2f and Rbf can co-immunoprecipitate with ORC through interactions that are independent of their respective DNA-binding activities (Bosco, 2001).

What is the functional relevance of this E2f-Rbf-ORC complex? One possible mechanism is that E2f-Rbf helps localize ORC to E2f-binding sites near the chorion replication origin. Another possibility is that ORC localization to the chorion replication origin is independent of its interaction with E2f-Rbf, and instead E2f-Rbf when bound next to an origin regulates replication initiation through its interaction with ORC. ORC binds the critical amplification control element ACE3 in vivoat a specific time in follicle cell development (stages 10A and 10B). Using anti-ORC2 antiserum, ACE3 has been specifically enriched relative to a control locus that does not bind ORC and is not amplified by using chromatin immunoprecipitation (CHIP). Using CHIP it was asked whether E2f also could be shown to localize specifically to ACE3 in vivo. Stabilization of protein-DNA interactions in live tissue is achieved by formaldehyde crosslinking. Subsequent CHIP enriches for specific trans-factors that are bound to genomic loci. The relative amounts of these loci are quantified by polymerase chain reaction (PCR). Sequence analysis reveals that there are several potential E2f-binding sites within 2.5 kilobases (kb) of ACE3. Using anti-E2f antibodies, it has been shown that ACE3 DNA is enriched ~15-fold relative to the rosy locus in stage 10 egg-chambers. Similarly, anti-ORC2 antibodies also enriched ACE3 DNA ~20-fold relative to the rosy locus. Thus, both E2f and ORC localize to ACE3 when amplification is occurring, and E2f binding is limited to sequences immediately adjacent to ACE3. This observation is consistent with E2f-Rbf functioning at replication origins and possibly controlling ORC activity (Bosco, 2001).

Previous work has shown that mutant follicle cells producing this truncated E2fi2 protein specifically localize ORC to the amplification regions as in wild type, but that such cells have elevated levels of ACE3 amplification. This elevated level of amplification is probably due to extra rounds of origin initiation events, suggesting that both E2f and Rbf have a negative regulatory function in origin firing during amplification. The DNA-binding domain of the truncated E2fi2 protein might be sufficient to localize ORC, if it could still interact with ORC. Therefore, whether or not the truncated E2fi2 protein complexes with ORC was tested. Immunoprecipitation experiments show that truncated E2fi2 does not interact with ORC. This means that the C-terminal domain of E2f is necessary for its interaction with ORC, and possibly requires Rbf to mediate this interaction. In contrast to the stated hypothesis, however, localization of ORC to the amplification region does not require a physical complex with E2f (Bosco, 2001).

Thus, the Drosophila E2f-Rbf complex functions during S phase, specifically to regulate DNA replication initiation at origins. It is thought that DP-E2f-Rbf are bound near ORC at the amplification origin and regulate initiation by forming a complex with ORC. Although E2f does not direct ORC binding, it restricts its activity through Rbf. Five lines of evidence form the basis for this model: (1) reduced levels of Rbf result in increased gene amplification levels and genomic replication without measurable effects on transcription of E2f target genes; (2) a complex of dDP-E2f-Rbf-ORC is present in ovary extracts; (3) this complex is independent of DNA binding; (4) truncation of the C terminus of E2f eliminates this complex, and (5) in this truncation mutant, ORC is localized but increased amplification occurs. The mechanism by which the dDP-E2f-Rbf complex limits replication initiation at the chorion locus remains to be determined. It is possible that the dDP-E2f-Rbf proteins inhibit the activity of the ORC subunits through a physical interaction. Alternatively, E2f-Rbf might inhibit loading of other replication factors at origins, such as MCM proteins. Finally, Rbf might alter the local chromatin configuration, for example by histone deacetylation, and thereby affect origin firing. Although ORC does not need to be in the E2f-Rbf complex to bind specifically to the chorion replication origin, a mutation in the DNA-binding domain of E2f does result in loss of ORC localization in the follicle cells. This observation needs to be evaluated in the context of the result that ORC is localized in the E2fi2 mutant, in which the truncated E2f protein is able to bind DNA but does not complex with ORC. Thus, DNA binding by E2f seems to be a prerequisite for ORC localization, but ORC localization does not require complex formation with E2f. This may be because when E2f is not bound to the chorion region, E2f2 can bind to sites at ACE3 normally occupied by E2f, and E2f2-Rbf may repel ORC and preclude localization or antagonize ORC binding activity (Bosco, 2001).

The Rbf mutant provides insights into the controls leading to the cessation of the endo cell-cycle during follicle cell development. Both the female-sterile Rbf mutant and the dDP female-sterile mutant show inappropriate continuation of the endo cell cycle beyond stage 10 of egg-chamber development. In contrast, an ectopic S phase does not occur in either of the female-sterile E2f mutants. Like the dDPa1 mutant, the Rbf120a/Rbf14 mutant is expected to have effects on both E2f-Rbf and E2f2-Rbf complexes. Thus, it seems that DP-E2f2-Rbf is needed to exit endo cycles, whereas DP-E2f-Rbf is involved more directly in regulating ORC and gene amplification. Identification of mutations in E2f2 will permit direct analysis of the roles of E2f2 in the endo cell cycle and amplification. Although it has not been shown whether any other specific replication origins may be regulated in this manner, the E2f-Rbf-ORC complex has been found in embryonic extracts, indicating that E2f-Rbf may be a general repressor of replication origins in embryonic tissues. Notably, a region between the DmPolalpha and E2f genes, containing several known E2f-binding sites, has been identified as a replication initiation region. Human RB (and associated HDACs) co-immunolocalize to BrdU foci in early S phase of primary cells, suggesting that RB may have a role in replication initiation. This observation is consistent with the model that suggests that Drosophila E2F1-Rbf localizes to replication origins and regulates ORC activity through a direct protein-protein interaction. It will be of great value to determine whether mammalian E2F-RB complexes can interact with ORC. Such an interaction would allow for a better understanding of how E2F and RB function to regulate DNA replication and cell proliferation during tumor progression (Bosco, 2001).

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. A functional recombinant Drosophila ORC has been reconstituted and activities of the wild-type and several mutant ORC variants have been compared. Drosophila ORC is an ATPase, and the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA+ nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. Evidence is provided that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features between Drosophila and budding yeast ORC complexes (Chesnokov, 2001).

Six different mutant complexes and wild-type recombinant ORC were prepared. For each case, simultaneous expression of the wild-type or mutant genes in a baculovirus expression system resulted in complexes that could be purified to homogeneity through four chromatographic steps, and the mutant complexes assembled and exhibited no chromatographic differences during the purification. In a final step, the pooled peak fractions were subjected to glycerol-gradient sedimentation (Chesnokov, 2001).

The best understood functions of the yeast ORC are its DNA-binding and ATP hydrolysis functions. The bulk of recombinant (or purified embryonic) Drosophila ORC DNA binding activity is nonspecific and ATP-independent. However, this ATP-independent DNA binding activity can be titrated away with sufficient amount of carrier DNA when the carrier DNA is in a range 50-100 molar excess to the probe DNA. At physiologically relevant ATP concentrations (10 microM to 1 mM), the wild-type ORC binds to DNA 10-50-fold better than either the ORC1A or ORC1B mutant complex. Mutations in either the Walker A or B motif of ORC4 or the Walker A motif of ORC5 have no effect on the formation of ATP-dependent DNA-protein complex. These experiments supports the idea that the recombinant Drosophila ORC, like the recombinant S. cerevisiae homolog, requires only the ORC1 component of the complex to bind ATP for tight DNA interactions. However, the complex missing the ORC6 subunit does not form an ATP-dependent DNA-protein complex (Chesnokov, 2001).

Kinetic analysis of ATP hydrolysis with multiple independent wild-type (wt) ORC preparations shows a Km of 1.92 µM and a Vmax of 0.4 mol ATP hydrolyzed per min per mol of complex. Binding to DNA has a small (2-fold) but measurable effect on slowing the rate of ATP hydrolysis by ORC. In these experiments, ATP was not limiting, the mutant ORI complexed to DNA was titrated to its maximal effect. In the absence of any carrier DNA, the saturation is reached at an approximate 2.5-fold molar excess of DNA to ORC. Complexes harboring similar mutations in either ORC4 or ORC5 hydrolyzes ATP with equivalent kinetics to wild type, all displaying Km values and Vmax within the experimental error range of wild type. Consistent with the DNA-binding experiments, the ATP-hydrolysis rate for these mutant complexes is slowed by DNA similar to the effect observed for the wild-type ORC. In contrast, ORC1A or ORC1B mutants have severely crippled enzymatic activity, too close to background to measure any kinetic parameters. The ORC-6 complex is able to hydrolyze ATP at reduced levels, but this activity is unaffected by DNA, consistent with the finding that ORC6 is critical for formation of an ATP-dependent ternary complex (Chesnokov, 2001).

Chromatin binding assays were performed by using both mutant and wt ORC in extracts depleted of membranes. For these experiments Drosophila preblastula embryo extracts were immunodepleted of ORC by using antibody raised against ORC2 and ORC6. The effectiveness of immunodepletion was verified by immunoblotting. Demembranated sperm chromatin was added to the depleted extracts, and the binding activities of mutant and wild-type recombinant DmORC were compared with the endogenous Drosophila ORC. Treatment of the extracts with Apyrase abolishes ORC-chromatin binding, thus it is inferred that the binding process requires ATP. Endogenous ATP levels (which are estimated to be at 30-50 µM) were relied upon to mediate tight chromatin binding. Proteins associated with the chromosomes are separated from the unbound proteins by sedimentation. The results obtained via this assay parallel those obtained by the gel-shift experiments. Recombinant wt ORC, ORC4A, ORC4B, and ORC5A complexes associate with the chromatin with apparently the same efficiency as does endogenous protein, whereas the ORC1A, ORC1B, and ORC 6 complexes are severely crippled (Chesnokov, 2001).

Two independent measures of DNA replication competence were used for accessing the abilities of the mutant complexes to restore activity to depleted extracts. In the first assay, labeled precursor incorporation into high molecular DNA was detected by autoradiography of gels after electrophoresis or in a second assay after CsCl density gradient separation of DNA that was replicated in extracts with the density label precursor BrdUrd. As anticipated from the DNA and chromatin binding results, the ORC1A, ORC1B, and ORCdelta6 complexes were essentially inactive by at least 10-20-fold below the activity of wt recombinant ORC in restoring replication to the extracts. The ORC4A, ORC4B, and ORC5A mutants were effective in reconstitution but were in multiple experiments between 50% and 90% of wild-type complex (Chesnokov, 2001).

It has been concluded that the bulk of the subunits of the Drosophila ORC biochemically behave as a complex. ORC2 antibodies were used to track ORC in fractions from 0-12-h embryo extracts after gel-filtration chromatography. Two broad zones containing ORC were found. The highest apparent molecular weight fractions containing all ORC subunits were pooled and purified. A smaller complex was also detected that was apparently without ORC-1. However, when following ORC6 using ORC6-specific antibodies, a pool of ORC6 devoid of other ORC subunits is detected. No other ORC subunits were found in a form unassociated with other ORC proteins. It is estimated that this free pool is at least one-half of the total ORC6 protein present in these extracts. Given the important role that Drosophila ORC6 plays in cell-free replication and the other activities of ORC, it was of interest to ask whether this separate pool of ORC6 is localized with the other ORC subunits in the cell (Chesnokov, 2001).

Transient ectopic expression of ORC1 or ORC2 GFP-fusion proteins in cultured cells shows a distinct nuclear localization; in unexpected contrast, the GFP-ORC6 fusion protein was found both in the nucleus and cytoplasm. The ORC6 cytoplasmic signal seems to be closely associated, in various focal planes, with the cytoplasmic membranes. These experiments rely on overexpression: this issue was probed further by direct immunofluorescence of endogenous levels of the ORC proteins in Drosophila embryos. Before the onset of cellularization, ORC6 protein localizes only with ORC2 in the nuclear space of both interphase and mitotic cells. However, after cellularization, ORC6 seems to localize in the cytoplasm and nucleus. The signals for ORC6 can be blocked by preincubating the affinity-purified antibodies with recombinant ORC6 proteins and are clearly distinct from the ORC2 pattern. Further work will be required to judge whether the cytoplasmic pool of ORC6 is truly membrane associated, but it is worth noting that the carboxyl terminus of Drosophila ORC6 contains a predicted leucine-zipper region that could be involved in mediating multiple heterologous protein-protein interactions (Chesnokov, 2001).

An important finding of this study is that the Drosophila ORC complex likely uses mechanisms for binding DNA that are similar to those reported for the budding yeast homolog. Of the three potential ATP binding proteins in ORC, only ORC1 seems to be critical for establishing a tight ternary complex with DNA and for binding to chromatin. Similarly only mutations in the ATP binding domains of ORC1 critically affect a single round of DNA replication in cell-free extracts. Additional experimentation needs to be done to test the roles of the conserved domains in ORC4 and ORC5. Particularly intriguing is the wide conservation of the GKT (Walker A motif) and D (D/EE) (Walker B motif) in the ORC4 subunit. Such domains may be critical for recycling ORC for subsequent rounds of replication or for other activities of the complex in heterochromatin formation or putative check-point control. Drosophila ORC is an ATPase, and again ORC1 seems to play the critical role for ATP hydrolysis, since mutants in the putative ATPase domains of ORC4 and ORC5 do not affect the kinetic parameters of the mutant complex. Nevertheless, it is possible that in the presence of other bound factors, ATP binding or hydrolysis by the other subunits plays some critical role. ATP hydrolysis by any subunit does not seem important for DNA-binding activity. ADP could not mediate such a DNA-protein complex, and ATPgammaS is better at forming a ternary complex than ATP. X-ray crystallographic structure models for several AAA+ proteins have been solved, and a common fold has been observed. The crystal structure model of an archael Cdc6 ortholog was used as a guide for the ATP-binding structures of ORC1. In the nucleotide-binding domain of this protein family, both the GKT and the DE motifs contribute to nucleotide affinity. In fact, similar mutants in the amino-part of the Walker B motif of the S. cerevisiae ORC1 are defective for ATP binding, in contrast to mutations at the carboxyl end of the B motif that are competent for such activity. Moreover, the solvent-exposed surfaces present in these parts of the ORC1 protein may influence interactions with other partners, yielding a mutant complex with altered functions. These studies of the ATPase activity of DmORC indicate that turnover is slower when ORC is bound to DNA, but the effect is significantly less than that observed for the budding yeast complex. Divergence in the way in which these proteins interact with DNA is also highlighted by the critical role that the Drosophila ORC6 protein plays in ATP-dependent DNA binding. Perhaps, given the lack of amino acid homologies found between the ScORC6 and DmORC6 proteins, it is dangerous to consider each to be homologs (Chesnokov, 2001).

Overexpression of ORC1 trans-genes in Drosophila can alter DNA replication patterns. This overexpression leads to detectable levels of BrdUrd incorporation in normally quiescent cells or increased levels of replication in follicle cells normally amplifying the chorion genes. Similar ectopic expression of an ORC1A mutant (ORC1K604E) has no phenotype. The biochemical results with the ORC1A mutant K604A predict that their mutation might have a dominant negative effect on DNA replication in vivo. It is possible that the mutant gene would not be antimorphic by virtue of its not being able to compete with a wild-type ORC1 protein for assembly into complex. Leaving this point aside, one idea favored is that ORC1 is limiting for replication in some cellular environments and, for example, complexes containing solely ORC2-6 wait for ORC1 for activation. These pools may or may not be bound to chromosomal DNA. Recent work in mammalian systems indicates that ORC1 may be more loosely associated with chromatin than is ORC2. ORC2, presumably with some of the subunits, can be pelleted with the chromosomes. The results reveal that intact ORC needs ATP and functional ORC1 to bind tightly to chromatin. Are all of these data compatible, assuming a conservation in basic binding properties for ORC between mammals and Drosophila? Perhaps, in the absence of ORC1 other subunits mediate another sort of chromatin association. More complex notions are possible, including the interaction of unknown chromatin binding proteins that serve to tether a complex lacking ORC1 to the ori sites (Chesnokov, 2001).

It is suggested that ORC6 is another subunit that may play important and perhaps dynamic roles in regulating replication activity. The data show that ORC6 is an essential component of the complex per se and may be directly involved in DNA binding and other replication functions or needed for proper ORC assembly. In H. sapiens extracts, ORC6 is not found associated with other ORC subunits, but when expressed in the baculovirus system with the other ORC genes, the protein does join a six-subunit complex. The high levels of free ORC6 in embryonic and cultured cell extracts is intriguing. A considerable fraction of this pool as judged by cytological methods is cytoplasmic, and the protein is perhaps associated with or proximal to the cytoplasmic membranes. It is possible that this localization enables ORC6 to participate in functions unrelated to DNA replication per se, as has been suggested for the 'latheo' gene product, which is ORC3. Latheo seems to be involved in ion transport at neuromuscular junctions. Data now exist for both the budding yeast and for the Drosophila ORC, which directly indicate that all of the subunits are critical for DNA replication function, and complex models involving traffic of subsets of ORC subunits can be the subject of future work (Chesnokov, 2001).

Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is constant and ORCs are bound constitutively to replication origins. Replication is regulated by the recruitment of additional factors such as CDC6. ORC components are widely conserved, and it generally has been assumed that they are also stable factors bound to origins throughout the cell cycle. In this report, it is shown that the level of the ORC1 subunit changes dramatically throughout Drosophila development. The accumulation of ORC1 is regulated by E2F-dependent transcription. In embryos, ORC1 accumulates preferentially in proliferating cells. In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with high levels in late G1 and S phase. In the ovary, the sub-nuclear distribution of ORC1 shifts during a developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion gene clusters. Furthermore, it has been found that overexpression of ORC1 alters the pattern of DNA synthesis in the eye disc and the ovary. Thus, replication origin activity appears to be governed in part by the level of ORC1 in Drosophila (Asano, 1999)

Late in embryonic development, most cells enter an extended quiescent period, resuming DNA synthesis upon hatching. However, replication persists in three tissues (brain, ventral nerve cord, anterior- and posterior-midgut), and the mRNAs of E2F-regulated genes (such as Ribonucleotide reductase, RNR2) accumulate in these tissues. To determine whether transcription of ORC1 is regulated by E2F, the distribution of ORC1 mRNA was examined in wild-type and E2F- embryos by in situ hybridization. The distributions of ORC1 and RNR2 mRNAs are essentially the same in wild-type embryos at stage 13. Moreover, accumulation of either RNR2 or ORC1 mRNA is largely dependent on E2F function at this stage of development. Thus, transcription of ORC1 is E2F-dependent in the embryo. It was next determined whether E2F regulates ORC1 transcription in imaginal disc cells, which have canonical four-phase cell cycles. Accumulation of ORC1 mRNA is induced following overexpression of E2F (~4-fold). By comparison, accumulation of three other E2F-regulated mRNAs - PCNA, RNR1 and RNR2 - is induced to a similar extent in these experiments (Asano, 1999).

To determine whether the regulation described above is mediated by the direct action of E2F, the ORC1 promoter was isolated. Within the 400 nt upstream of the major transcriptional start site, four candidate E2F binding sites with similarity to the canonical site in the adenovirus E2 promoter (TTTCGCGC) were identified by inspection, two at approximately -340 nt and two overlapping sites at -13 nt. Characterization of other E2F-responsive promoters has shown that binding sites close to the transcriptional start site frequently play a predominant role in regulation, and thus a focus was placed on the overlapping sites at -13. Drosophila E2F has been shown to bind to the ORC1 promoter just upstream of the start site of transcription. To test the role of these E2F sites in vivo, transcriptional reporter genes were prepared in which either the wild-type ORC1 promoter or a mutant derivative bearing substitutions within the proximal E2F binding sites drives the expression of a cDNA encoding an unstable Ftz-GFP-Myc tag fusion protein. Activity of the ORC1 promoter is dependent on the integrity of the E2F binding sites at -13 nt. In flies bearing the wild-type promoter construct, fusion protein is detectable in cells throughout most regions of the imaginal discs. However, in flies bearing a mutant promoter construct, essentially no fusion protein is detectable in any of the imaginal discs. These observations suggest that E2F acts directly by binding to the ORC1 promoter and stimulating transcription. Furthermore, the spatiotemporal pattern of ORC1 promoter activation within two specialized groups of cells in the eye and wing imaginal discs supports the idea that E2F couples transcription of ORC1 to cell cycle progression (Asano, 1999).

During the third larval instar, a developmentally regulated cell cycle transition takes place as a wave of differentiation sweeps across the eye imaginal disc. The wave front is marked by the morphogenetic furrow. During differentiation, four regions can be identified: (1) anterior to the morphogenetic furrow (including the antennal disc), undifferentiated cells cycle asynchronously; (2) as they enter the furrow, cells arrest in an extended G1 phase; (3) immediately posterior to the furrow, some cells are recruited into ommatidial pre-clusters and begin neural differentiation while others synchronously enter S phase, and (4) posterior to this synchronous wave of S phase, most cells cease cycling and terminally differentiate. The pattern of ORC1 promoter activity in the eye imaginal disc suggests that it is turned on late in G1, near the G1-S boundary. In particular, the ORC1 promoter is activated in a random pattern among the asynchronous cells in the anterior region of the disc: turned off as cells enter the morphogenetic furrow and G1, turned on in cells as they emerge from the furrow late in G1 phase, and then turned off in the quiescent cells in the posterior region of the eye. Another developmentally programmed cell cycle arrest has recently been described in the wing imaginal disc. At the dorsoventral boundary of the disc, Notch and wingless signaling establish a zone of non-proliferating cells (ZNC) in which no S phase is detectable. Cells throughout the posterior ZNC and in the center of the anterior ZNC arrest late in G1, at a point when expression of Cyclin E can drive them into S; flanking cells in the anterior ZNC arrest in G2. Among the cells of the ZNC, the ORC1 promoter is active only in G1-arrested cells and not in those arrested in G2. These observations support the idea that activation of E2F in G1 stimulates transcription of ORC1 in a variety of cell types (Asano, 1999).

In Drosophila, many genes have been shown to be transcriptionally regulated by E2F during the G1-S transition. These include Cyclin E, RNR, Polalpha , PCNA, MCM2 and MCM3 . However, only in the case of Cyclin E has it been shown that protein levels are cell cycle regulated, presumably at least in part as a consequence of E2F action. In the other cases, either the protein distribution has not been reported or the protein level is constant throughout the cell cycle. Therefore, to determine whether the level of ORC1 is modulated as a result of E2F-dependent regulation, antibodies were prepared that specifically recognize the protein, and its distribution was examined in embryos and imaginal discs. Antisera were prepared by immunizing animals with glutathione S-transferase (GST) fusion proteins bearing three different portions of ORC1. The distribution of ORC1 was examined during embryonic development. The first 13 nuclear division cycles that occur in a syncitium are parasynchronous. However, upon formation of the cellular blastoderm and the onset of gastrulation, this synchrony breaks down. Subsequent cell divisions are synchronous within cohorts of adjacent cells, but cell cycles within adjacent 'mitotic domains' are out of register. During the first 13 synchronous cell cycles, maternally synthesized ORC1 is uniformly distributed among the embryonic nuclei. However, coincident with the formation of the cellular blastoderm and the onset of gastrulation, the ORC1 distribution changes dramatically, such that different nuclei contain very different levels of protein. For example, mesodermal precursors along the ventral midline, which comprise one of the mitotic domains, accumulate relatively high levels of protein at the onset of gastrulation. During germ band extension, ORC1 levels are highest among the mitotically active neuroblasts and in domains of epidermal precursor cells. Later, in stage 13 embryos, when most cells in the embryo are cell cycle arrested, ORC1 accumulates preferentially in cells of the nervous system and midgut that continue to cycle. In summary, the level of ORC1 in the Drosophila embryo is not constant as is the case in S.cerevisiae. Instead, the protein is developmentally regulated such that high levels of protein are found in proliferating cells (Asano, 1999).

Two lines of evidence suggest that E2F-dependent transcriptional regulation is responsible (at least in part) for this differential accumulation of ORC1. (1) In stage 13 E2F- embryos, essentially no ORC1 is detectable. (Analysis of E2F-dependence in earlier embryonic stages is confounded by the maternal supply of E2F.) (2) The pattern of ORC1 accumulation is mirrored by the patterns of ORC1 promoter activity and ORC1 mRNA accumulation during embryonic development. Therefore, it is concluded that E2F-dependent transcriptional regulation, at least in part, couples ORC1 accumulation to proliferation. The distribution of ORC1 was examined in eye-antennal imaginal discs, where a developmentally regulated cell cycle transition takes place as the morphogenetic furrow sweeps across the disc. The level of ORC1 changes dramatically during this G1-S transition. The level of protein initially is low among the G1-arrested cells in the furrow. As cells emerge from the furrow late in G1, the level of ORC1 rises. Following the completion of S phase, ORC1 levels fall, returning to the basal level seen in the furrow. Two additional observations suggest that these changes in ORC1 levels are not peculiar to cells in and around the morphogenetic furrow. (1) Cells with high and low levels of ORC1 are randomly interspersed in the anterior region of the eye disc and the antennal disc where cells cycle asynchronously. (2) Within the ZNC of the wing imaginal disc, cells arrested in G1 accumulate high levels of ORC1, whereas G2-arrested cells do not. High levels of ORC1 accumulate in cells 3-4 rows to the posterior of the furrow and S phase begins in cells 5-6 rows to the posterior. Since a new row of cells emerges from the furrow every 1.5 h, this suggests that ORC1 accumulates ~1.5-3 h before the onset of S phase. ORC1 levels fall only after the completion of S phase. In summary, the level of ORC1 is cell cycle regulated, with peak accumulation during late G1 and throughout S phase. Further overexpression studies show that the abundance of ORC1 regulates DNA synthesis. In wild-type discs, cells within the morphogenetic furrow never incorporate BrdU, and cells in the posterior region of the disc do so only rarely at this stage of development. However, in HS-ORC1 discs, some cells in both of these regions incorporate BrdU and thus appear to have entered S phase either prematurely or inappropriately. Ectopic ORC1 has no effect on either the onset or duration of the synchronous S phase among cells that emerge from the furrow. Nor does ectopic ORC1 have any noticeable effect on the proliferation of imaginal discs, the intensity of labeling at different BrdU concentrations or the growth rate of transgenic animals. Furthermore, the observation that endogenous ORC1 levels rise in anticipation of entry into S phase in the eye disc is consistent with the idea that high levels of ORC1 promote DNA synthesis rather than the opposite. As is the case in the imaginal discs, activity of the ORC1 promoter is E2F-dependent in the ovary (Asano, 1999).

Visualization of replication initiation and elongation in Drosophila

Chorion gene amplification in the ovaries of Drosophila is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, it was found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2-7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. This system was used to demonstrate that the pre-replication complex component, Double-parked protein/Cdt1, is not only necessary for proper MCM2-7 localization, but, unexpectedly, is present during elongation (Claycomb, 2002).

Three independent lines of evidence are presented that initiation and the bulk of elongation at a chorion amplicon occur during two separate developmental periods. (1) Deconvolution microscopy shows that ORC and BrdU initially colocalize at origins and then diverge, since ORC is lost in stage 11 and BrdU resolves into a double bar structure. (2) Elongation factors PCNA and MCM2-7 follow the same pattern as BrdU, resolving from foci early in amplification to a double bar structure by stage 12 to 13. (3) Quantitative realtime PCR shows a peak increase in DNA copy number at the origins by stage 11, with increases in flanking sequences becoming substantial in stages 12 and 13. Thus initiation ends by stage 11, and during stages 12 and 13 only the existing forks progress outward. Furthermore, these observations led to the unanticipated conclusion that DUP/Cdt1 travels with replication forks (Claycomb, 2002).

The realtime PCR and immunofluorescence data are remarkably consistent. (1) Both methods restrict initiation to stages 10B and 11 of oogenesis, and elongation to stages 12 and 13. Between stages 10B and 11, the maximum fold amplification was detected at amplification control element (ACE) on third chromosome (ACE3) by realtime PCR, ORC localized to origins, and the deconvolution showed a maximum increase in bar length. During stages 12 and 13, increases in fold amplification were detected only proximal and distal to ACE3, and ORC no longer localized to origins, whereas BrdU incorporation resolved into the double bar structure. (2) The distances of fork movement are consistent. Deconvolution measurements predicted that forks were maximally 30 +/- 3 kb apart in stage 10B, and this correlates with the 40-kb span of peak copy number detected by realtime PCR. In stage 11, forks were measured to have progressed across a 55 +/- 13-kb region by deconvolution and across a 45-kb region by realtime PCR. By stage 13, deconvolution showed that replication forks were maximally separated by 74 +/- 7 kb, whereas realtime PCR measured a 75-kb span (Claycomb, 2002).

The quantitative analysis of the amplification gradient provides insight into mechanisms affecting fork movement and termination and suggests that an onionskin structure impedes fork movement. The maximal rate of fork movement during amplification has been calculated to be 90 bp/min on average. In comparison, replication forks in the polytene larval salivary glands travel at ~300 bp/min (Steinemann, 1981), whereas rates of fork movement in both diploid Drosophila cell culture and embryo syncytial divisions are ~2.6 kb/min. From these rates, it seems that polyteny hinders replication fork movement, an effect even more pronounced in amplification, given that the chorion cluster has a rate of fork movement three times less than polytene salivary glands. The fact that by stage 13 there is a gradient of copy number, and not a plateau, further demonstrates the inefficiency of fork movement along the chorion cluster (Claycomb, 2002).

There do not seem to be specific termination sites to stop forks either along or at the ends of the chorion region, but fork movement may display some sequence or chromatin preference. The gradient of decreasing copy number implies that forks stop at a range of sites, because the presence of specific termination points along the region would be expected to cause steep drops in copy number. Despite this lack of specific termination sites, during stages 12 and 13 a greater increase is seen in copy number to one side of ACE3, and it was often observe by immunofluorescence that one of the two bars is shorter. This suggests that the sequence or chromatin structure to the other side of ACE3 hinders fork movement, and as fewer forks move out, less BrdU incorporation occurs and a shorter bar results (Claycomb, 2002).

These studies highlight the complex regulation of chorion gene amplification. How are the number of origin firings restricted to the proper developmental time? It is known that the number of rounds of origin firing at the chorion amplicons is limited by the action of Rb, E2F1, and DP. Perhaps Dup and MCM2-7 are also a part of this regulation, with origins firing only when MCM2-7 are properly loaded. It will also be interesting to decipher the regulation of Dup/Cdt1 during amplification. Recent studies have demonstrated that a Drosophila homologue of the metazoan re-replication inhibitor, Geminin, exists and interacts biochemically and genetically with Dup/Cdt1. Female-sterile mutations in geminin result in increased BrdU incorporation during amplification, raising the possibility that Geminin acts on DUP/Cdt1 at the chorion loci to limit origin firing. In addition to permitting the delineation of the regulatory circuitry controlling origin firing, the ability to developmentally distinguish initiation from elongation provides a powerful tool for the analysis of the properties of metazoan replication factors in vivo (Claycomb, 2002).

Drosophila Mcm10 interacts with members of the prereplication complex and is required for proper chromosome condensation

Mcm10 is required for the initiation of DNA replication in Saccharomyces cerevisiae. MCM10 from Drosophila has been cloned; it complements a ScMCM10 null mutant. Moreover, Mcm10 interacts with key members of the prereplication complex: Mcm2, Double parked (Dup or Cdt1), and Orc2. Interactions were also detected between Mcm10 and itself, Cdc45, and Hp1. RNAi depletion of Orc2 and Mcm10 in KC cells results in loss of DNA content. Furthermore, depletion of Mcm10, Cdc45, Mcm2, Mcm5, and Orc2, respectively, results in aberrant chromosome condensation. The condensation defects observed resemble previously published reports for Orc2, Orc5, and Mcm4 mutants. These results strengthen and extend the argument that the processes of chromatin condensation and DNA replication are linked (Christensen, 2003).

Mcm10 was first identified in S. cerevisiae as defective in S-phase progression and subsequently was shown to be defective in the maintenance of minichromosomes. Work on Mcm10 in S. cerevisiae has revealed that Mcm10 interacts with members of the pre-RC and is required for efficient initiation of DNA replication. Mutants of Mcm10 exhibit pausing of replication forks, suggesting a role for Mcm10 in elongation. Chromatin fractionation experiments show that Mcm10 is constitutively bound to chromatin. Analysis in human has shown that Mcm10 interacts with Orc2, is phosphorylated, and is degraded by an ubiquitin-dependent pathway during the cell cycle. Recent work in Xenopus demonstrates that Mcm10 is required for replication, is dependent on Mcm2-7 for association with the origin, and is necessary for recruitment of Cdc45 (Christensen, 2003 and references therein).

The Drosophila homolog of Mcm10 was first identified by Izumi (2000). The study used the predicted Drosophila Mcm10 to identify homologous human ESTs. The Drosophila Mcm10, known as CG9241, maps to the 2nd chromosome and cytologically to 39B1. Advantage was take of known EST sequences to design primers to amplify MCM10 from cDNA isolated from Drosophila ovary tissue. Sequencing of the resulting clone revealed that Drosophila Mcm10 is a 776 amino acid protein with a predicted molecular mass of 86.5 kDa. Overall, it shares similarity to Human (32.1%), Xenopus (30.5%), Arabidopsis thaliana (29.7%), Caenorhabditis elegans (26.2%), S. cerevisiae (24.1%), and Schizosaccharomyces pombe (23.1%). Alignments of the conserved regions of human, Xenopus, Drosophila, and S. cerevisiae reveal that Drosophila Mcm10 shares a conserved central core and a signature zinc finger motif. In addition, there is a high degree of regional conservation between Mcm10 of Drosophila and higher eukaryotes that points to the fact that studies in Drosophila will have significant bearing on those in Xenopus and human (Christensen, 2003).

Mcm10 has been shown in yeast to interact with all members of the Mcm2-7 family except for the notable exception of Mcm5. In addition, human Mcm10 interacts with Orc2. To investigate which members of the pre-RC interact with Mcm10 in Drosophila coimmunoprecipitation experiments were performed. A stable KC cell line containing Mcm10::GFP was induced or not induced with Cu2+ and cells were harvested, processed, and immunoprecipitated with anti-GFP. Interactions with Mcm10-GFP are detected with Mcm2, Orc2, and the endogenous Mcm10, consistent with the two-hybrid studies. Also probed and shown positive for interactions are Dup, Cdc45, and Hp1. No interaction is detected with Mcm5, and Orc1. Immunoprecipitations were also performed using antibodies to Cdc45, Dup, Mcm2, Orc2, Mcm5, Orc1, and Hp1, respectively, in embryo cell extracts. Similar to the coimmunoprecipitation results with Mcm10-GFP, in all but Mcm5 and Orc1, Mcm10 is detected (Christensen, 2003).

In the absence of a known Drosophila mutant for Mcm10, RNAi was used to determine the function of Mcm10 in Drosophila. RNAi involves addition of dsRNA specific to the mRNA sequence of the target gene. RNAi acts to deplete the mRNA of the target species. The result is that the protein of interest is specifically depleted from cells at a rate corresponding to the inherent stability of the protein. RNAi has been demonstrated as an effective tool in Drosophila tissue culture for determining gene function. In this analysis, KC cells at low densities were inoculated with specific dsRNA and collected over a 5-d period for immunoblot analysis. Over the course of the experiment, cells grew from low to high densities. Low densities corresponded to cell cycle time of ~22 h, and the apparent cell cycle lengthens to 40+ h at high density as cells begin to exit into G0. Mcm10, Cdc45, Mcm2, Mcm5, and Orc2 are all efficiently depleted from KC cells upon addition of specific dsRNA (Christensen, 2003).

Mcm10 and Ccd45 are particularly sensitive to RNAi treatments because both are depleted by 48 h and are undetectable by Western blot. The rapid depletion is indicative of either inherent instability of these proteins or regulation via proteolysis. Mcm2 and Mcm5 show depletion of the bulk of the protein by 48 h but both remain at low levels. In contrast, Orc2 is slowly depleted over the time course compared with the others. This observation is supported by the fact that null mutants for Orc2 in Drosophila persist until third instar, presumably due to the stability of maternal deposits (Christensen, 2003).

Several interesting observations are apparent from these experiments. First, both Mcm10 and Cdc45 exhibit sensitivity to exit into G0 and/or increasing cell densities as shown by the fact that both are reduced in the nonspecific treatment. This is in contrast to Mcm2, Mcm5, and Orc2, which all show increases correlating with increased cell densities and are seen to accumulate as cells exit into G0 and/or increase in density as measured over this time course. These observations suggest that overall stability of Mcm10 and Cdc45 may be regulated as a function of the cell cycle, regulated in relation to cell densities, or a combination of both. In contrast, overall stability of Mcm2, Mcm5, and Orc2 does not seem to be regulated with respect to the cell cycle and/or increased cell densities. The relatively short-lived Drosophila Mcm10 is consistent with observations reported for the human Mcm10. HsMcm10 protein levels are regulated by both phosphorylation- and ubiquitin-dependent proteolysis during late M and early G1 phase. In contrast, S. cerevisiae Mcm10 has been shown to be present at constant levels throughout the cell cycle (Christensen, 2003).

At the outset, one would predict that depletion of proteins required for initiation of DNA replication would have dire consequences for cell growth. Cell growth of KC cells treated with dsRNA specific to Mcm10 and Orc2 was assayed. Over the course of 6 d the growth of cells depleted of Orc2 and Mcm10 seemed unaffected. One could argue that because of the short time assayed and the fact that apparent cell division slows as cell density increases that one would not be able to detect a significant decrease in growth. To address this, a more rigorous test was performed. Cells were diluted every 3 d to keep them at densities that allow logarithmic growth rate. Concurrently, cells were inoculated with specific dsRNA when diluted to ensure that translation of the specific genes did not recover from the initial RNAi treatment. Cell divisions were quantified and cumulative cell divisions calculated over an 18-d period. Cells well past depletion of any detectable Orc2 or Mcm10 divided at wild-type rates. The same phenomenon was observed for depletion of Mcm2, Mcm5, and Cdc45 (Christensen, 2003).

The fact that long-term depletion of Orc2 to <10% had no effect on cell division rates demanded further investigation into how Orc2 depletion was tolerated. Given that KC cells are aneuploidy, it seems reasonable that these cells could tolerate some degree of chromosome loss. Depletion of Orc2, which is involved in initiation of DNA replication, and depletion of Mcm10, which has been shown in yeast to be required for initiation of DNA replication, may have consequences for the DNA content of KC cells (Christensen, 2003).

To investigate possible DNA content effects in Drosophila KC cells depleted of Orc2 and Mcm10 (both positive regulators of DNA replication) were analyzed by FACS. Cells were inoculated with specific dsRNA and maintained in the continual presence of dsRNA and harvested after 10 cell cycles. FACS analysis indicates that both Orc2-depleted and Mcm10-depleted cells show a loss of DNA content compared with controls. This suggests that depletion of Orc2 and Mcm10 both have consequences on the ability of cells to maintain DNA content that may result from decreases in the efficiency of DNA replication or chromosome stability (Christensen, 2003).

RNAi is specific to the target protein. However, because depletion of a particular protein does not occur in a vacuum but rather in a network of interactions, Mcm10 stability was examined in cells depleted of other proteins. KC cells were diluted in the presence of specific dsRNA for ~10 cell cycles. Cells were harvested, lysed, and whole cell extracts were loaded onto SDS-PAGE gels. Blots were then probed for Mcm10, Cdc45, Mcm2, Mcm5, Orc2, Dock, and lamin. Total Mcm10 protein levels are reduced in KC cells depleted of Mcm2, Orc2, and to a lesser extent Mcm5. Cdc45 levels are reduced when Mcm10, Mcm2, and Mcm5 are depleted. Mcm2 levels are slightly reduced when Orc2 is depleted. Last, Orc2 levels are relatively unchanged in all but the specific treatment (Christensen, 2003).

Depletion of Mcm10 or Cdc45 results in strikingly similar defects in chromosome morphology. The condensation defects apparent in both depletions consist of similar 'dumbbell' shaped chromosomes. Sister chromatid separation is observed in both treatments with a higher frequency observed in cells depleted of Cdc45. Chromosome fragmentation in Mcm10 and Cdc45 depletions is observed at levels no higher than that of wild type and is likely a consequence of specimen preparation. The fact that these defects are so similar combined with the findings that these two proteins have been shown to interact supports the supposition that these two proteins function in concert in the same pathway (Christensen, 2003).

Depletion of Mcm2, Mcm5, or Orc2 results in a high degree of fragmentation in addition to lateral condensation defects suggestive of incomplete DNA synthesis and subsequent unchecked chromosome separation. Mcm2 depletion demonstrates the most severe defect with no wild-type figures found and an overwhelming percentage in class III, the most severe class. Sister chromatid separation is noted in all three treatments but is more prevalent in Mcm2-depleted cells. The small discrepancies between Mcm2 and Mcm5 depletions with respect to severity may represent merely different stability levels of the protein since Mcm2 is depleted more rapidly and thoroughly by RNAi (Christensen, 2003).

Thus Drosophila Mcm10 biochemically interacts with components of the pre-RC, including Mcm2, Orc2, and Dup, and with itself. These interactions further the argument that Mcm10 is present in the pre-RC before the transition to the pre-IC and that interaction of Mcm10 with its various partners may be mediated by a Mcm10 multimer Christensen, 2003).

This study presents the first evidence for an interaction between Mcm10 and Double parked (Dup), the Drosophila homolog of Cdt1. This interaction is of particular interest in light of the observation that Cdt1 is required for loading of the MCM complex and is a target for geminin regulation. These and other studies suggest that Cdt1 interacts with the ORC and MCM complexes. In this context, Mcm10 may be interacting directly with Dup or indirectly through ORC or the MCMs to facilitate steps in pre-RC assembly (Christensen, 2003 and references therein).

Mcm10 interaction with Cdc45 has been implied previously in Xenopus where it has been shown that Cdc45 requires Mcm10 for origin binding. In addition, it has been shown that S. cerevisiae Mcm10 genetically interacts with Cdc45. The experiments presented in this study extend the interaction between Cdc45 and Mcm10 to Drosophila and support the hypothesis that stabilization of Cdc45 binding to origins may occur via a direct or indirect interaction with Mcm10 (Christensen, 2003).

Heterochromatin protein 1 (Hp1) has been shown to interact with members of the ORC complex. Interaction with Mcm10 suggests that, like Orc2, Mcm10 may be associated with heterochromatin to facilitate the role of Hp1 in heterochromatin formation, maintenance, transcriptional repression, or epigenetic inheritance. An interaction between Mcm10 and Hp1 may point to a trend that more members of the pre-RC are involved in heterochromatin formation. Indeed, this process may be a fundamental function of prereplication proteins. The involvement of ORC in establishing silencing at the mating type loci in yeast has been pointed to as an analog to the function of ORC in establishment of heterochromatin in Drosophila. In fact, the function of ORC in silencing and heterochromatin formation may be the most conserved aspect of ORC function. Drosophila Orc2, for example, is able to complement the silencing defect of a mutant Orc2 allele in yeast, but it is unable to complement the replication defect. In addition to ORC, Cdc45 and Mcm2 have been implicated in yeast to be involved in chromatin formation. The implication that Mcm10 is involved in formation of heterochromatin in Drosophila by virtue of an interaction with both Orc2 and Hp1 raises the tantalizing possibility that these dynamic properties of chromatin may require not only ORC function but also a number of other prereplication proteins as well (Christensen, 2003).

Key members of the pre-RC and pre-IC are effectively and specifically depleted from KC cells by RNAi. Although deficiencies in DNA replication were not directly tested, replication must still be occurring at a level sufficient to maintain growth rates under long-term depletion of Mcm10 and Orc2. Precedence for this phenomenon has been reported in human HCT116 colon carcinoma cells where 10% of the wild-type level of Orc2 are sufficient to sustain normal chromosomal replication. Maintenance of DNA content at levels that permit cell viability may be due to these proteins being required in very small amounts combined with the hypothesis that few origins are required to replicate the genome. The results suggest that RNAi generates severe hypomorphs for protein depletions and not total depletion (Christensen, 2003).

No explanation is available for the mechanism that selectively retains a complement of chromosomes that ensures viability as a result of reduced DNA replication. Precedence for ploidy effects by depletion of proteins by RNAi in Drosophila SD2 cells has recently been reported for both geminin and Dup. This study reports that depletion of geminin, a negative regulator of DNA replication, resulted in an increase in DNA content. In contrast, depletion of Dup, a positive regulator of DNA replication, results in loss of DNA content. Defects in the growth of these cells were not reported (Christensen, 2003).

Protein levels of certain members of the pre-RC may be coupled. These observations could be due to at least three different factors or combinations of factors. (1) Depletion of one protein results in transcriptional repression of another either directly or indirectly. (2) Some or all of these depletions result in cell cycle defects that have consequences for other proteins, although cell cycle length is unchanged. (3) Interactions between proteins are required for stability. In other words, proteins are stable in a complex but not individually. An example of this is the coupling of Cdt1 and geminin protein levels observed when geminin is depleted from tissue culture. Depleting cyclin A also results in a corresponding decrease in cyclin B protein levels. Another case of association-dependent stabilization is the destruction of Cdc6 when removed from chromatin and association with the pre-RC (Christensen, 2003).

This is the first report of a possible role for Mcm10, Mcm2, Mcm5, and Cdc45, respectively, in proper condensation of chromosomes. This study demonstrates the utility of RNAi in tissue culture cells for assaying chromosomal condensation defects. Indeed, it points the way to analysis of other known replication proteins for which mutants do not yet exist with respect to functions in condensation. An interesting point to consider is that the process of chromosome condensation may be more sensitive to the dosage of these proteins than is DNA replication, because cells remain viable over long-term depletion. These observations raise the intriguing possibility that the bulk of these proteins in cells may function in chromosome condensation pathways and not overtly participate in the initiation of DNA replication. Alternatively, the depletion of these proteins may simply reduce the number of initiation sites along the chromosome, resulting in fewer replication foci. This reduction in foci may have direct mechanistic consequences for condensation. A third possibility is that depletion of replication initiation factors in S phase may force cells to enter mitosis prematurely, resulting in aberrant chromosome condensation. Indeed, depletion of Cdt1/Dup protein first shown in S. pombe and more recently shown in Drosophila results in chromosome condensation without DNA replication and thereby bypassing S phase. It is not believed that the third possibility is a likely explanation because no dramatic loss of cell viability or a change in cell cycle length was observed as expected of mitosis without S phase. Determining whether these proteins are directly linked to condensation or are merely linked via DNA replication is a question that remains to be answered (Christensen, 2003).

It is becoming increasingly clear that proteins involved in DNA replication are necessary for establishment of proper chromosomal condensation. There is some debate as to whether the defects observed are due to the specific functions of individual proteins or are a general function of compromised DNA replication. The addition of Mcm10, Mcm2, Mcm5, and Cdc45 to the repertoire of replication proteins required for proper chromosomal condensation lends support to the hypothesis that DNA replication and condensation are generally linked (Christensen, 2003).

What is the mechanism by which replication is linked to condensation? At the outset, it seem reasonable that organization of chromatin would happen in concert with replication. Spatially and temporally separating the processes would seem to be both inefficient and problematic with respect to entanglement of DNA and nuclear organization. A simple mechanism for linkage of replication to condensation has been put forth that suggests that the density of replication initiation along a chromosome and the resulting DNA replication foci has impact, on a primary level, on the lateral condensation of a metaphase chromosome. This hypothesis fits very well with the 'dumbbell' lateral condensation defects observed when replication proteins are depleted (Christensen, 2003 and references therein).

The simple mechanistic model probably has relevance to the linkage of condensation to DNA replication but may not provide a complete picture as to the role of pre-RC proteins in this process. There are several observations that speak to the possibility that the proteins of the pre-RC have roles outside of DNA replication. A comparison of two recent studies in yeast looking at global binding of prereplication complexes and global origin usage reveals that there are 30% less active origins compared with those predicted by binding of pre-RC proteins. These observations point to the fact that sites not used for initiation are occupied by members of the pre-RC, leaving open the possibility for some functional role for these assemblies in chromatin condensation (Christensen, 2003).

This study has presented evidence for the conservation of Mcm10 function from Drosophila to S. cerevisiae. Drosophila Mcm10 interacts with known members of the pre-RC, consistent with a role in the assembly of the pre-RC. Moreover, Mcm10 interacts with Cdc45, suggesting that Drosophila Mcm10 may also participate in the transition to the pre-IC. Further evidence for a role of Mcm10 in DNA replication comes out of the observation that depletion of Mcm10 by RNAi, similar to that of Orc2, resulted in a loss of DNA content. Mcm10 is also required for proper chromosome condensation. This role may be facilitated by an interaction with Hp1 (Christensen, 2003).

Cul4 and DDB1 regulate Orc2 localization, BrdU incorporation and Dup stability during gene amplification in Drosophila follicle cells

In higher eukaryotes, the pre-replication complex (pre-RC) component Cdt1 is the major regulator in licensing control for DNA replication. The Cul4-DDB1-based ubiquitin ligase mediates Cdt1 ubiquitylation for subsequent proteolysis. During the initiation of chorion gene amplification, Double-parked (Dup), the Drosophila ortholog of Cdt1, is restricted to chorion gene foci. This study found that Dup accumulated in nuclei in Cul4 mutant follicle cells, and the accumulation was less prominent in DDB1 (piccolo) mutant cells. Loss of Cul4 or DDB1 activity in follicle cells also compromised chorion gene amplification and induced ectopic genomic DNA replication. The focal localization of Orc2, a subunit of the origin recognition complex, is frequently absent in Cul4 mutant follicle cells. Therefore, Cul4 and DDB1 have differential functions during chorion gene amplification (Lin, 2009).

In this study, Cul4 and DDB1 mutants were isolated which were larval lethal with growth arrest in the second instar stage, similarly to previous results (Hu, 2008). It was further shown that Cul4 mutant clones in developing wing discs were defective in proliferation and had a reduced number of S-phase cells. To focus on the role of Cul4 during DNA replication and bypass the requirement of Cul4 in G1-S transition, mutant follicle cells were analyzed during gene amplification stages in follicle cells. It was shown that the Dup protein level and Orc2 focal localization are regulated by Cul4 and, differentially, by DDB1. In addition, BrdU focal patterns are defective in Cul4 and DDB1 mutant follicle cells (Lin, 2009).

Previous studies have shown the replication-dependent degradation of human and Xenopus Cdt1 by the Cul4-DDB1 E3 ligase, and the replication-coupled recruitment of the DDB1 to chromatin in Xenopus cells. This study has shown the requirement of Cul4 for the suppression of Dup protein levels during gene amplification in Drosophila follicle cells. Comparison of Dup nuclear accumulation in Cul4 and DDB1 mutant follicle cells, however, reveals substantial differences. Almost all Cul4 mutant cells, except those undergoing apoptosis, accumulated Dup in the nucleoplasm in stage 10B, consistent with Cul4 being a dedicated component of the E3 ligase in promoting Cdt1 degradation. By contrast, accumulation of Dup levels was observed in much smaller fractions of two DDB1 alleles analyzed. These analyses have not excluded the involvement of DDB1 in downregulating Dup protein levels. Compensatory or parallel Cul4-mediated Dup degradation pathways might be present in addition to the Cul4/DDB1-mediated Dup degradation. In agreement with this speculation, a recent study has shown that nuclear accumulation of cyclin D1 during the S phase promotes human Cdt1 stabilization and triggers DNA re-replication. This Cdt1 stabilization could be suppressed by the overexpression of Cul4A and Cul4B but not DDB1 or Cdt2, also an adaptor for the Cul4 ligases, implying the involvement of other adaptors in mediating the Cul4-dependent degradation of Cdt1 (Lin, 2009).

This study found that BrdU incorporation at chorion gene amplification foci was reduced or even absent in about half of Cul4G1-3 mutant follicle cells, if apoptotic cells that cannot be scored for their capability in BrdU incorporation were excluded. Similarly, 40% of Cul4G1-3 mutant cells displayed ectopic BrdU incorporation (the abnormal genomic replication group). These cells were not scored for their BrdU incorporation at focal sites because of overall strong nuclear signals. Therefore, the effect of Cul4 on the chorion gene amplification might be underestimated in this Cul4-null allele. Using the Cul4 hypomorphic allele KG02900 in which both the fractions of apoptotic cells and abnormally BrdU-incorporated cells were reduced, the combined percentages for reduced and absent BrdU incorporation combined at chorion gene amplification foci reached more than 70%. DDB15-1 mutant follicle cells also displayed a severe phenotype in the BrdU incorporation assay. When apoptotic cells were disregarded, cells with a reduction or absence of BrdU incorporation accounted for more than 60% of DDB15-1 mutant cells. Therefore, these BrdU incorporation analyses lend support to the notion that certain processes in chorion gene amplification require both Cul4 and DDB1 (Lin, 2009).

These BrdU foci represent DNA amplification of chorion genes within 100 kb of origins, and the phenotype of absence or reduction in BrdU incorporation could reflect a failure in the initiation of DNA replication, reduced processivity in DNA synthesis or fewer rounds of gene amplification. To further support the idea that Cul4 is involved in gene amplification, advantage was taken of the dominant-negative Cul4KR mutant in which the neddylation site has been mutated. Acute expression of Cul4KR suppressed BrdU incorporation in almost all follicle cells. When assayed by quantification PCR, gene amplification at chorion foci was strongly suppressed, supporting a role of Cul4 in the chorion gene amplification process (Lin, 2009).

Abnormal genomic replication, as inferred from ectopic BrdU incorporation, was observed in Cul4 mutant follicle cells in both Cul4 mutant alleles tested. The percentage of cells with such a defect was reduced in the cells with the hypomorphic mutant allele, indicating that the low level of Cul4 activity partially suppresses this defect. Abnormal genomic replication was also detected in DDB1 mutant follicle cells with a lower frequency than in Cul4-null mutants. The phenotype of ectopic genomic replication is less likely to be a retarded developmental process in the previous endocycle stage, because these cells with ectopic BrdU signals show normal DNA contents as estimated by Hoechst staining. Ectopic genomic replication might require some prerequisite steps in DNA replication, such as Orc2 localization at replication origins, which is defective in Cul4 mutant cells, thus blocking abnormal DNA replication throughout the genome (Lin, 2009).

The localization of Orc2, a component of the pre-RC, at chorion gene foci was examined during gene amplification. Mutations in Cul4 caused reduced or no Orc2 localization at gene amplification foci, a prominent phenotype in both G1-3 (59%) and KG02900 alleles (60%) when apoptotic cells were discounted. Failure of proper Orc2 focal localization might represent defects in the initial loading of Orc2 or the maintenance of Orc2 localization at amplification foci. Such Orc2 localization defects were not prominent in DDB1 mutant follicle cells (Lin, 2009).

Consequently, defective Orc2 localization at regular gene amplification foci might evoke ectopic genomic replication in Cul4 mutant follicle cells. Upon co-labeling for Orc2 and BrdU in Cul4 mutant cells, the absence of or reduction in Orc2 signals was found in conjunction with a reduction in BrdU incorporation at gene amplification loci or with abnormal BrdU incorporation throughout the genome. In some cells, normal Orc2 loading was accompanied with abnormal BrdU incorporation. The decoupling of both phenotypes therefore suggests that Cul4 functions distinctively in Orc2 localization and in suppression of abnormal BrdU incorporation during chorion gene amplification (Lin, 2009).

Many interesting questions remain to be answered regarding how genetic loci are selected for amplification, how the pre-RC is assembled only in specific loci and how other genomic regions are kept silent. Previous evidence suggests that high transcriptional activity of specific loci controls replication origin firing during the gene amplification stage. Mutants for transcription factors such as E2f2, Dp, Rbf, Myb and Mip130 show increased mRNA and protein levels of replication factors, such as components of the Orc and MCM complexes, and ectopic genomic replication during the gene amplification stage. Mutant follicle cells for Cul4 displayed both Orc2 localization defects and abnormal genomic replication, implying that Cul4 is probably involved in both processes by modulating the transcriptional activity in DNA replication. Interestingly, a recent study suggests that Cul4 targets degradation of the transcriptional activator E2F1 during S phase. However, Drosophila E2F1 proteins became abundant in the nucleus and rich at ACE3 origin DNA during gene amplification. How this developmental regulation of E2F is involved in Cul4 activity needs further investigation. Some studies suggest that Cul4 functions in histone modification and heterochromatin maintenance. It is speculated that the Cul4 E3 complex also functions in regulating Orc2 origin localization through a local remodeling of the chromatin structure on ACE3 and Ori-β (Lin, 2009).



ORC2 is most abundant in embryos 0 to 4 hours old and constitutes 0.01% of the total protein (Gossen, 1995). This distribution correlates with the observation that major waves of rapid cell division occur in embryos during the first 4 hours of development. In rapid nuclear cleavages, the high rate of DNA synthesis is achieved in part by a frequency of dectectable active origins that is 100 times the frequency seen in cultured Drosophil cells lines (Gossen, 1995).

Requirements for DNA replication origin function

The developmentally regulated amplification of the Drosophila third chromosome chorion gene locus requires multiple chromosomal elements. Amplification control element third chromosome (ACE3) appears to function as a replicator, in that it is required in cis for the activity of nearby DNA replication origin(s). Ori-ß is the major origin in the locus, and is a sequence-specific element that is sufficient for high-level amplification in combination with ACE3. Sequence requirements for amplification were examined using a transgenic construct that was buffered from chromosomal position effects by flanking insulator elements. The parent construct supported 18- to 20-fold amplification, and contained the 320 bp ACE3, the ~1.2 kb S18 chorion gene and the 840 bp ori-ß. Deletion mapping of ACE3 revealed that an evolutionarily conserved 142 bp core sequence functions in amplification in this context. Several deletions had quantitative effects, suggesting that multiple, partially redundant elements comprise ACE3. S. cerevisiae ARS1 origin sequences could not substitute for ori-ß, thereby confirming the sequence specificity of ori-ß. Deletion mapping of ori-ß identified two required components: a 140 bp 5' element and a 226 bp A/T-rich 3' element called the ß-region that has significant homology to ACE3. Antibody to the origin recognition complex subunit 2 (ORC2) recognizes large foci that localize to the endogenous chorion gene loci and to active transgenic constructs at the beginning of amplification. Mutations in Orc2 itself, or the amplification trans regulator satin eliminated the ORC2 foci. By contrast, with a null mutation of chiffon (dbf4-like) that eliminates amplification, diffuse ORC2 staining was still present, but failed to localize into foci. The data suggest a novel function for the Dbf4-like Chiffon protein in ORC localization. Chromosomal position effects that eliminated amplification of transgenic constructs also eliminate foci formation. However, use of the buffered vector allowed amplification of transgenic constructs to occur in the absence of detectable foci formation. Taken together, the data suggest a model in which ACE3 and ori-ß nucleate the formation of an ORC2-containing chromatin structure that spreads along the chromosome in a mechanism dependent upon Chiffon (Zhang, 2004).

The use of insulator elements, the suppressor of Hairy-wing protein binding sites [su(Hw)BSs], protects transgenic chorion gene constructs from chromosomal position effects and allows for detailed analysis of sequence requirements for amplification. The ACE3 replicator and ori-ß origin elements are necessary for efficient amplification. A construct containing only the 320 bp ACE3 and the 840 bp ori-ß ('Small Parent' or SP) demonstrates that these elements are also sufficient for amplification; however, the levels of amplification are moderate and are subject to significant chromosomal position effects even in the presence of the flanking insulator elements. In the BP construct, the 320 bp ACE3 and the 840 bp ori-ß are in their normal context, i.e., spaced by the ~1.2 kb S18 chorion gene, and these sequences supported efficient amplification (~20 fold) with minimal position effects. For this reason, the BP construct was chosen for detailed analysis of ACE3 and ori-ß sequence requirements. Evolutionarily conserved core sequences were found to be sufficient for the majority of ACE3 activity. Deletion of the less conserved 5' and 3' flanking sequences within ACE3 had quantitative effects, suggesting that multiple, partially redundant elements comprise ACE3. These results and conclusions are analogous to those from a previous study of ACE3 sequence requirements done in the context of a larger, unbuffered construct. No deletion of a subset of ACE3 sequences reduced amplification to the extent of a deletion of all of ACE3. The sequence requirements for ACE3 function in amplification defined in this study correlate well with the sequence requirements previously defined for ORC binding in vitro. The central region of ACE3, corresponding to the evolutionarily conserved sequences, is most crucial for ORC binding, while the 5' and 3' flanking regions within ACE3 stimulates ORC binding. Taken together, the data suggest that the multiple, partially redundant elements that comprise ACE3 are ORC binding sites, and that one crucial function of ACE3 in amplification is to bind ORC (Zhang, 2004).

Recently a protein complex containing Drosophila Myb, p120 and three other proteins was found to bind to both ACE3 and ori-ß sequences, and Myb was found to be required in trans for amplification. Both Myb and p120 are capable of DNA binding on their own, and have binding sites that overlap with the essential core region of ACE3. There are two Myb consensus binding sites (positions 121 to 127 and 137 to 142) and three p120 binding regions (27 to 56, 89 to 105 and 184 to 216) in ACE3 element. Small (30-40 bp) deletions that removed one of Myb consensus binding sites or one of the p120-binding sites in the core region of ACE3 had negative effects on amplification in the context of the BP construct. Taken together, these data suggest that another function of the conserved core region sequences of ACE3 is to bind the Myb complex (Zhang, 2004).

Two-dimensional gel analyses of the endogenous third chromosome chorion gene locus demonstrate that the majority (70%-80%) of initiations occur in a region containing the ori-ß element. In 2D gel analysis of the BP construct, abundant initiation events, as indicated by bubble structures, were associated with the ori-ß element while no initiations could be detected for ACE3. To begin to examine the sequence requirements for ori-ß function, ori-ß was substituted by either the entire 193 bp S. cerevisiae ARS1 origin sequence, or the 20 bp B2 element from ARS1, which is a putative DNA unwinding element. No activity in supporting amplification was detected for either fragment, indicating that ori-ß is not simply an A/T-rich or easily unwound sequence. Deletion mapping suggests two sub-components of ori-ß: an essential 5' 140 bp region that is not particularly A/T-rich, and the 226 bp A/T-rich ß region. The 366 bp fragment containing both regions is sufficient for the majority of ori-ß activity. In addition the 3' most 140 bp of the starting 840 bp ori-ß fragment may have a small stimulatory effect. The portion of the alpha region in ACE3 and the ß region in ori-ß are each A/T-rich and internally repetitive, and have some sequence homology with each other. A large fragment containing the ß-region can bind ORC in vitro. Therefore, it is hypothesized that, like the sequences in ACE3, one required function of the ß region in ori-ß is to bind ORC (Zhang, 2004).

A similar organization has been identified for a developmentally regulated origin in another dipteran fly, Sciara coprophila. In Sciara larvae, the salivary gland cells amplify several loci containing putative pupal case genes, resulting in chromosomal DNA 'puffs'. The ori II/9A DNA replication initiation site has been mapped to the nucleotide level and has similarities to the yeast ARS. Drosophila ORC has been shown (Bielinsky, 2001) to bind to an 80 bp region adjacent to this replication start site (Zhang, 2004).

Analysis of trans-acting gene mutations confirms the intimate association between amplification initiation and the formation of a large focus of ORC2 localization at amplifying chromosomal loci. Mutations in k43 (Orc2) itself, or the newly identified trans-regulatory gene satin, eliminated ORC2 antibody staining and focus formation. Null mutations of chiffon, a dbf4-like gene, completely eliminate amplification. In chiffon-null mutant follicle cells, diffuse ORC2 staining was still present in the nucleus, but it failed to localize into foci at stage 10A. A similar phenotype had previously been observed for mutations in the amplification trans-regulators dDP (a subunit of E2F) and Rbf. A role for chiffon in ORC localization was surprising given the well-characterized order of events known for other organisms. In S. cerevisiae and Xenopus in vitro systems, ORC is bound at origins and is required for the subsequent binding of Dbf4 and its catalytic subunit CDC7, which is one of the last events before origin firing. The data suggest two possible models for the role of chiffon in ORC2 focus formation during amplification. In the first model, Chiffon protein would bind first to the chorion gene sequences, either directly or more likely via an interaction with another DNA-binding protein, since the Chiffon sequence suggests no obvious DNA-binding motifs. Chiffon would then recruit Drosophila ORC2 to the DNA. This model seems unlikely given the opposite order of events observed in yeast and in Xenopus in vitro systems. In the second and favored model, a relatively small amount of ORC binds first to the chorion gene loci, most probably to the conserved core sequences in ACE3 and the ß region in ori-ß. Chiffon protein would then interact with the ORC complex(es) and catalyze the further binding of large amounts of ORC to generate the dramatic foci observed upon staining with ORC2 antibody. A mechanism is envisioned in which the alpha and ß regions nucleate ORC binding, and then through a process dependent upon Chiffon, an ORC-containing chromatin structure spreads along the chromosome to form the dramatic foci. This model is appealing in that it provides a way for ACE3 and ori-ß to interact and form a chromosomal domain activated for DNA initiation events. Previous data have indicated that ACE3 and ori-ß interact during amplification in a way that can be blocked by an intervening insulator element. Moreover, analysis of the endogenous locus indicates that ACE3 is required for the activation of multiple origins spread throughout a chromosomal domain containing the chorion gene cluster. This model is testable in that it predicts that the insulators would form a boundary for this ORC-containing chromatin structure (Zhang, 2004).

The possibility cannot be ruled out that chiffon is not the true Dbf4 homolog in Drosophila, but this appears unlikely. Chiffon shows conservation with Dbf4 homologs from all other species in the key ORC-binding domain (called CDDN2) and the CDC7-binding domain (called CDDN1). Moreover, there is no other gene in the Drosophila genome with detectable homology to Dbf4. However, Chiffon contains an additional large C-terminal protein domain present only in Dbf4 homologs from closely related species, such as Medfly and mosquito. It is speculated that this C-terminal domain may play a specific role in chorion gene amplification. Further experiments will be required to determine if a role in ORC localization is a characteristic of all Dbf4 family members, or whether this represents a function unique to the large chiffon protein (Zhang, 2004).

Consistent with the correlation between ORC2 focus formation and amplification initiation, dramatic ORC2 foci can form at the sites of amplifying transgenic chorion gene constructs. It was therefore surprising that in no cases were foci observed at the sites of actively amplifying BP constructs. This is despite the fact that amplification was readily observed at these sites by BrdU incorporation. One possible explanation might be the moderate amplification level of BP (18- to 20-fold). However, the YES-3.8S construct amplifies to similar levels as BP, and an extra ORC2 focus was observed for every line. In addition multimers of ACE3 with very low amplification levels are capable of creating additional ORC2 foci. Therefore, the lack of focus formation with BP is not simply due to its moderate amplification level, but must reflect the specific sequence content or arrangement in BP. The lack of focus formation in BP is also not simply due to the presence of flanking insulator elements; the YES-3.8S construct contains the same flanking insulator elements. The data suggest two non-exclusive possibilities. The first is that the difference is due to the fact that BP contains less extensive chorion gene sequences than YES-3.8S. Although deletion of these sequences has no significant effect on amplification level, it may be that redundant ORC binding sites have been deleted, thereby dramatically reducing visible focus formation. The second possibility is that the relevant difference is the amount of sequence present inside the insulators. BP contains only 2.4 kb between the insulators, whereas Yes-3.8 contains 9 kb. If the insulators limit the size of the domain in which an ORC containing chromatin structure can spread from ACE3 and/or ori-ß, then the small size of this domain in BP may not create a visible focus. In this model, the insulators would have two significant effects on amplification: they would prevent the spread of negative chromatin structures into the bounded region and thereby prevent negative chromosomal position effects; and they would limit the ORC containing chromatin structure and initiation activity to the bounded region. These models should be testable in the future by CHIP analysis of chromatin structures associated with chorion gene sequences and transgenic constructs. It will be of interest in the future to determine if su(Hw)BS insulators or other types of insulators are involved in organizing the endogenous chorion gene locus and the rest of the genome into domains of DNA replication activity (Zhang, 2004).

Effects of Mutation or Deletion

The k43 gene was identified in the Nusslein-Volhard laboratory by means of a single female-sterile mutant allele causing abnormally thin and fragile chorions (eggshells). The chorion defect is due to greatly reduced levels of chorion gene amplification during oogenesis. During Drosophila oogenesis the chorion gene loci are normally amplified about 80 fold through repeated initiation of DNA replication at one or a small number of origins located within each of the two chorion gene clusters. Chorion gene amplification allows the follicle cells to produce the large amounts of protein required in a short time for normal chorion synthesis. The process of reinitiation is likely to require at least some of the general DNA replication factors involved in the function of other chromosomal DNA replication origins, at other stages of the life cycle. In addition, there must be amplification-specific activities that limit the process to the follicle cells, and to only the DNA replication origins associated with the chorion gene clusters. k43 gene function is required for normal chorion gene amplification. The recessive female-sterile allele k43fs293 greatly reduces chorion gene amplification, resulting in underproduction of chorion proteins, and thus defective chorions. The null phenotype of k43 is lethal to late larvae, with defects limited to dividing diploid cells. Homozygous larvae exhibit small or missing imaginal discs, and low mitotic index. The few mitotic cells present exhibit fragmented chromosomes and irregular chromatin condensation. Taken together, the k43 mutant phenotypes suggest a role for k43 in chromosomal DNA replication. In fact, k43 encodes the Drosophila homolog of yeast orc2 (Landis, 1997).

The accurate duplication and packaging of the genome is an absolute prerequisite to the segregation of chromosomes in mitosis. To understand the process of cell-cycle chromosome dynamics further, the first detailed characterization has been performed of a mutation affecting mitotic chromosome condensation in a metazoan. A combined genetic and cytological approach in Drosophila complements and extends existing work employing yeast genetics and Xenopus in vitro extract systems to characterize higher-order chromosome structure and function. Two alleles of the ORC2 gene were found to cause death late in larval development, with defects in cell-cycle progression (delays in S-phase entry and metaphase exit) and chromosome condensation in mitosis. During S-phase progression in wild-type cells, euchromatin replicates early and heterochromatin replicates late. Both alleles disrupt the normal pattern of chromosomal replication, with some euchromatic regions replicating even later than heterochromatin. Mitotic chromosomes were irregularly condensed, with the abnormally late replicating regions of euchromatin exhibiting the greatest problems in mitotic condensation. The results not only reveal novel functions for ORC2 in chromosome architecture in metazoans, they also suggest that the correct timing of DNA replication may be essential for the assembly of chromatin that is fully competent to undergo mitotic condensation (Loupart, 2000).

The phenotype reported here is specific to mutations in ORC2, and not observed generally for defects in other replication proteins. Mutations have also been analyzed in DmRfc4, subunit 4 of replication factor C (important for loading proliferating cell nuclear antigen (PCNA) to allow processive replication). Although mitotic defects are observed, they differ from the ORC2 phenotype described here (resulting in either prematurely condensed chromosome-like figures or premature sister chromatid separation), and are primarily due to defective checkpoint control. The localization of Rfc4 during mitosis is also different from that of ORC2, since the protein does not appear to rebind chromatin during anaphase. Thus, the ORC2 phenotype is specific to this protein, and not a general consequence of inhibiting proteins essential for replication. Mitotic phenotypes may exist for other mutations in replication proteins. Proliferation defects and decreased BrdU incorporation have been reported for Drosophila ORC3, MCM2 and MCM4 mutants, but the phenotype of mitotic cells has not yet been described for any of these mutations. Neuroblasts of PCNA mutants do not exhibit mitotic abnormalities (Loupart, 2000).

The intense ORC2 accumulation on mitotic chromosomes in late anaphase and telophase is striking, and similar to the observed localization of ORC1 in Xenopus cultured cells. Drosophila ORC2 is strongly localized to the centromeres of metaphase and anaphase chromosomes of early syncytial Drosophila embryos and physically associates with the heterochromatin-binding protein, HP-1. There is a low, but detectable, concentration of ORC2 on pericentric regions of anaphase chromosomes in cultured cells. These differences in ORC concentration on centromeres could be attributable to the inherent biological differences of the two stages of Drosophila development: the rapid S/M cycling occurring in embryos versus the slower cell cycles with Gap phases of neuroblasts. If ORC2 is retained more strongly at centromeric regions during embryogenesis, ORC may aid in the establishment of heterochromatin, which occurs concomitant with cellularization in Drosophila embryos. Clearly, additional information will be gleaned from the analysis of diploid and polyploid chromosomes from the DmORC3 mutant (Loupart, 2000).

Delay in entering S phase is accompanied by temporal disruption of replication. The reduced frequency of BrdU incorporation and increased occurrence of cells with a single centrosome both indicate an early cell-cycle delay in ORC2 mutant neuroblasts. If the ORC complex is unstable when ORC2 is mutated, then assembly of the pre-RC may be affected and the time taken to enter S phase prolonged. However, at least some ORC2 mutant neuroblasts enter S phase, albeit with slow progression. Detailed analysis of the mitotic chromosomes following S phase in the presence of BrdU highlights significant changes to the replication timing of at least some regions of euchromatin. Heterochromatin always replicates later than euchromatin in wild-type neuroblasts. In contrast, this temporal relationship is perturbed in the ORC2 mutants, such that some euchromatic regions became late replicating, even later than heterochromatin. This could be attributed to defects in RC formation and function in euchromatin (Loupart, 2000).

This effect on euchromatin could be the result of a higher affinity of ORC for heterochromatin. ORC2, at least, appears to be present on pericentric heterochromatin in early anaphase, but not on euchromatic arms until later in anaphase. Perhaps the interaction of ORC with HP-1 at heterochromatin stabilizes the assembly of RCs, ensuring the appropriate replication of these regions in the next cell cycle even when ORC2 function is compromised (Loupart, 2000).

It is concluded from two lines of evidence that abnormally late-replicating euchromatin is most affected in its ability to condense properly in Drosophila ORC2 mutant cells. (1) FISH with probes for pericentric repeats on the three large chromosomes shows that heterochromatin appears to condense normally. Therefore, it is likely that euchromatin is the major source of inadequately condensed chromatin. (2) Analysis of BrdU incorporation in the subsequent mitosis demonstrates that late-replicating euchromatin often exhibits condensation defects. DNA replication and the establishment of chromatid cohesion are intimately linked, elements of the latter process being laid down during S phase. Perhaps aspects of the condensation machinery are also 'templated' during S phase, and if replication is altered, then subsequent assembly may be affected (Loupart, 2000).

ORC may have a central role in organizing higher eukaryotic chromosomes. In S. cerevisiae, ORC has been proposed to act as a landing pad for assembly of the pre-RC and for transcriptional control. The results presented here raise the possibility that, in metazoans, the ORC landing pad may interact with many additional protein complexes, such as those necessary for cohesion, repair, condensation, and decatenation. A model is proposed to account for the mitotic chromosomal defects that were observed. This model takes into account the consequences of faulty ORC2 and pre-RC formation, with downstream effects on cohesion/condensation. It is postulated that, because of a higher affinity of ORC for heterochromatin, as pools of wild-type (maternal) protein dwindle during development, these will selectively be targeted to heterochromatin, enabling DNA replication with appropriate timing and facilitating cohesion and condensation. Regions of euchromatin deficient in ORC would not be replicated at the right time in S phase, and therefore lose the opportunity to assemble the chromatin structures required for metaphase chromosome condensation. ORC is depicted as a central landing pad in this model, though it could just as likely serve as the structural focus for the subsequent events of cohesion and condensation, each of which is dependent on the previous event occurring correctly. The status of cohesin and condensin subunits is currently being examined to ascertain how structural defects manifest themselves in cells exhibiting abnormal mitotic chromosome condensation (Loupart, 2000).

The origin recognition complex (ORC) is a six subunit complex required for eukaryotic DNA replication initiation and for silencing of the heterochromatic mating type loci in Saccharomyces cerevisiae. The discovery of the Drosophila ORC complex concentrated in the centric heterochromatin of mitotic cells in the early embryo and its interactions with heterochromatin protein 1 (HP-1) lead to a speculation that ORC may play some general role in chromosomal folding. To explore the role of ORC in chromosomal condensation, a mutant of subunit 5 of the Drosophila origin recognition complex (Orc5) has been identified and the phenotypes of both the Orc5 and the previously identified Orc2 mutant, k43 have been characterized. Both Orc mutants die at late larval stages and surprisingly, despite a reduced number of S-phase cells, an increased fraction of cells were also detected in mitosis. For this latter population of cells, Orc mutants arrest in a defective metaphase with shorter and thicker chromosomes that fail to align at the metaphase plate within a poorly assembled mitotic spindle. In addition, sister chromatid cohesion is frequently lost. PCNA and MCM4 mutants have similar to phenotypes Orc mutants. It is proposed that DNA replication defects trigger the mitotic arrest, due to the fact that frequent fragmentation is observed. Thus, cells have a mitotic checkpoint that senses chromosome integrity. These studies also suggest that the density of functional replication origins and completion of S phase are requirements for proper chromosomal condensation (Pflumm, 2001).

The analysis of Drosophila larvae with severely reduced ORC function has confirmed at a cellular level a role for ORC in cellular proliferation. At the simplest level, the vastly reduced BrdU labeling in the proliferative neural tissue of larvae homozygous for Orc2 or Orc5 mutations confirms a crucial role of ORC in cellular proliferation. At a higher magnification, the patchy and reduced levels of BrdU staining of chromosomes in Orc mutants is most consistent with a slowed S phase, with perhaps fewer origins used, and an ensuing mitotic arrest. It is suspected that this arrest is triggered by DNA damage sustained during a defective S phase. In addition, the presence of abnormally condensed chromosomes in Orc mutants, and in MCM4 and PCNA mutants has offered unexpected insight into a link between DNA replication and chromosome morphology (Pflumm, 2001).

It is emphasized that the severity of the chromosomal ORC phenotypes changes with developmental time, and it is postulated that this directly reflects the extent to which maternal pools have been depleted. In comparing the phenotypes of genes that encode different replication proteins, cells from the same tissue (larval neuroblasts) with equivalent defects in BrdU incorporation were compared. This, for example, precluded observations with a recessive lethal primase mutant, since no deficiency in replication levels was detected in the larval neuroblast (Pflumm, 2001).

One of the most striking observations from this study is that Orc and other mutants directly involved in DNA replication appear to be arrested at two stages of the cell cycle: in G1 and mitosis. The large majority of cells appear to be unable to enter S phase, since the severely reduced levels of BrdU incorporation observed probably reflect a G1 arrest. However, some mutant cells appear to accumulate in a state with many of the hallmarks of metaphase. Specifically, they arrest at a stage that lacks a nuclear membrane, with many characteristics of a spindle. Moreover, there is no evidence of anaphase bridges, characteristic of cells at more advanced stages of mitosis. Furthermore, in most cells, sister chromatids are evident, suggesting that cells have passed through S phase. However, the mitotic state observed in these mutants is defective because of the presence of abnormally condensed chromosomes, absence of a complete bipolar spindle and congression failure (Pflumm, 2001).

This defective metaphase arrest with abnormally condensed chromosomes was observed in all DNA replication mutants tested. Irrespective of the condensation mechanisms that are defective or inoperative in DNA replication mutants, broken chromosomes are probably the triggers that signal the mitotic arrest. Much of the data from genetic analysis of the structural maintenance of chromosome (SMC) protein complex suggests that incomplete condensation per se does not trigger mitotic arrest. SMC mutants proceed through the metaphase-anaphase transition normally with decondensed chromatin, but have chromosome segregation defects. In Drosophila, both barren (encoding a non-SMC protein likely associated with condensin) and gluon (an SMC4 homolog) mutants show chromosome defects with anaphase bridges. Instead, insufficient levels of DNA replication proteins may increase the levels of broken chromosomes and/or result in incomplete DNA replication, which would lead to a cell cycle arrest in late S phase. However, a number of such cells might break through such a checkpoint (Pflumm, 2001).

Cells transiently arrested in S/G2 because of incomplete replication or damaged DNA may then be halted in mitosis by checkpoints sensitive to chromosomal integrity. Treatment of wild-type embryos with the DNA synthesis inhibitor aphidicolin or irradiating embryos during mitosis results in a similar phenotype to that of DNA replication mutants: a metaphase arrest characterized by a poorly defined anastral mitotic spindle and chromosomes that do not align appropriately at the metaphase plate. This mitotic arrest is thought to operate with centrosome inactivation because mitotic spindles are anastral and the centrosomes are deficient for certain gamma-tubulin components. Indeed it is interesting that the gamma-tubulin mutant gammatub23C shows defective mitotic figures strikingly similar to those images reported here for Orc mutants. In addition, gamma-tubulin staining appears abnormal in all the DNA replication mutants tested in this study: gamma-tubulin staining often co-localizes with the chromosomes and is frequently fragmented into several pieces. Thus, centrosome inactivation may be part of an additional pathway serving to prevent damaged or incompletely replicated DNA from finishing mitosis (Pflumm, 2001).

If Drosophila cells have two mechanisms for monitoring damaged or incompletely replicated DNA caused by insufficient ORC levels or any protein involved in DNA replication, one would expect defects in the S phase checkpoint to increase the frequency of cells arrested in mitosis. grapes (grp) encodes for the Drosophila homolog of Chk1, a protein kinase required for a checkpoint-mediated cell cycle arrest triggered by DNA damage or incomplete DNA replication. Orc2;grp homozygous double mutants were compared with Orc2 homozygous larvae. No change in the frequency of mitotic cells was observed in such DAPI-stained mitotic spreads. This result may however reflect the multiple pathways that operate in S phase to monitor DNA damage in Drosophila. In S. cerevisiae, this multi-tiered monitoring of DNA damage, including a back-up checkpoint in mitosis may not be as effective. Yeast Orc mutants arrest in late stages of the cell cycle and it has been postulated that ORC might have an M phase function. However, recent work establishes that an orc2;rad9 double mutant (rad9 is a protein kinase required for the DNA damage checkpoint) does not arrest at such a discrete point (Pflumm, 2001 and references therein).

The most unanticipated aspect of this work is the impact of DNA replication mutations on the overall ultrastructure of mitotic chromosomes. The large majority of mitotic chromosomes were abnormally condensed and lacked distinct centromeres. The shorter and thicker appearance of these mitotic chromosomes suggests that an element of lateral condensation has been lost. The incomplete and abnormal chromosomal morphologies described here are interesting in light of present knowledge about mechanisms of chromosome condensation. The data add to the evidence that condensation is a multi-tiered process and not an all-or-none mechanism. Incomplete condensation in a DNA replication mutant strain may be an indirect consequence of S phase defects. For example, Orc deficiencies that lead to incomplete replication or damaged DNA may affect the levels of cyclin-dependent kinases, leading to reduced condensation. It is just as likely that replication plays a direct role in sculpting the morphology of the mitotic chromosome. For example, the density of active Orc loci on the chromatin fiber could influence the number of replication factories that would loop together such origins. Alternatively, bi-directional growing forks may be held together at a point and replicated DNA would be condensed as it is spooled through the machinery (Pflumm, 2001 and references therein).

The notion that each pair of replication forks that start at an origin remains attached has been proposed as a mechanism to help avoid entanglement of independently rotating forks. This idea has been further explored and it has been pointed out that such attachment would automatically lead to a length contraction of the chromosome. One of the predictions of this model is in striking concordance with actual observations. In DNA replication mutants, there is a lower density of replication centers and chromosomes are abnormally shorter and wider, as would be expected. Clearly, the number of such centers would affect the total level of chromosome condensation. Fewer replication centers predict much larger loops and a much reduced lateral chromosomal condensation (Pflumm, 2001).


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Origin recognition complex subunit 2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 February 2012

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