InteractiveFly: GeneBrief

Origin recognition complex subunit 6: Biological Overview | References


Gene name - Origin recognition complex subunit 6

Synonyms -

Cytological map position - 46B4-46B4

Function - DNA replication

Keywords - DNA-dependent DNA replication, mitotic cell cycle, regulation of cytoskeleton

Symbol - Orc6

FlyBase ID: FBgn0023180

Genetic map position - 2R:5,732,964..5,733,964 [-]

Classification - Origin recognition complex subunit 6

Cellular location - nuclear and cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Balasov, M., Akhmetova, K. and Chesnokov, I. (2015). Drosophila model of Meier-Gorlin syndrome based on the mutation in a conserved C-Terminal domain of Orc6. Am J Med Genet A. PubMed ID: 26139588
Summary:
Meier-Gorlin syndrome (MGS) is an autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears, and skeletal abnormalities. Patients with MGS often carry mutations in the genes encoding the components of the pre-replicative complex such as Origin Recognition Complex (ORC) subunits Orc1, Orc4, Orc6, and helicase loaders Cdt1 and Cdc6. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. Mutation in conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. In order to study the effects of MGS mutation in an animal model system. MGS mutation was introduced in Orc6, and a Drosophila model of MGS was established. Mutant flies die at third instar larval stage with abnormal chromosomes and DNA replication defects. The lethality can be rescued by elevated expression of mutant Orc6 protein. Rescued MGS flies are unable to fly and display multiple planar cell polarity defects.

BIOLOGICAL OVERVIEW

The origin recognition complex (ORC) is a 6-subunit complex required for the initiation of DNA replication in eukaryotic organisms. ORC is also involved in other cell functions. The smallest Drosophila ORC subunit, Orc6, is important for both DNA replication and cytokinesis. To study the role of Orc6 in vivo, the orc6 gene was deleted by imprecise excision of P element. Lethal alleles of orc6 are defective in DNA replication and also show abnormal chromosome condensation and segregation. The analysis of cells containing the orc6 deletion revealed that they arrest in both the G1 and mitotic stages of the cell cycle. Orc6 deletion can be rescued to viability by a full-length Orc6 transgene. The expression of mutant transgenes of Orc6 with deleted or mutated C-terminal domain results in a release of mutant cells from G1 arrest and restoration of DNA replication, indicating that the DNA replication function of Orc6 is associated with its N-terminal domain. However, these mutant cells accumulate at mitosis, suggesting that the C-terminal domain of Orc6 is important for the passage through the M phase. In a cross-species complementation experiment, the expression of human Orc6 in Drosophila Orc6 mutant cells rescued DNA replication, suggesting that this function of the protein is conserved among metazoans (Balasov, 2009).

The hexameric origin recognition complex (ORC) is an essential component for eukaryotic DNA replication. It was originally discovered in Saccharomyces cerevisiae, and subsequent studies both in yeast and in higher eukaryotes laid the foundation for understanding the functions of this important key initiation factor. ORC binds to origin sites in an ATP-dependent manner and directs the assembly of the prereplicative complex (pre-RC) at the origins. ORC subunits and/or complete ORC complexes have also been identified in many metazoan species, suggesting the existence of common mechanisms for the initiation of DNA replication in all eukaryotes. ORC genes are essential for cell survival. Mutational analysis of ORC-related genes in yeast and in higher eukaryotes reveals defects in DNA replication. In other studies, immunodepletion experiments using either Xenopus or Drosophila replication-competent extracts indicate an absolute requirement for ORC to initiate DNA replication. Acute depletion of ORC gene expression in human cells by RNAi resulted in cell cycle arrest. In addition to initiating DNA replication, ORC is involved in other functions that have been described in detail (Balasov, 2009).

The Orc6 protein is the least conserved of all ORC subunits. In S. cerevisiae, Orc6 is not important for DNA binding, but it is required for cell survival (Li, 1993; Newlon, 1993; Lee, 1997) and the maintenance of the pre-RC, specifically for recruitment of Cdt1 followed by MCM loading (Semple, 2006; Chen, 2007). Schizosaccharomyces pombe and metazoan Orc6 proteins are more homologous, similar in size, and considerably smaller than the S. cerevisiae Orc6. In Drosophila, Orc6 is essential for ORC-dependent DNA binding and DNA replication (Chesnoko, 2001; Balasov, 2007). In Xenopus and human systems Orc6 is less tightly associated with the core complex, and some of the published data suggest that Orc6 may not be important for these activities. This apparent inconsistency may reflect the difference in affinity of Orc6 for the core ORC1-5 complex in distant metazoan species. Drosophila Orc6 and human Orc6 also have a function in cytokinesis (Prasanth, 2003; Chesnokov, 2003; Chesnokov, 2009). This function in Drosophila is attributed to the C-terminal domain of Orc6 (Chesnokov, 2003). To study the Orc6 functions in a living organism, the Orc6-deletion mutant in Drosophila was generated and characterized. The mutant phenotypes associated with a lethal allele of the Drosophila orc6 gene were analyzed alone or with different versions of fly or human Orc6 rescue transgenes, gaining further insight into the roles Orc6 plays through the cell cycle in metazoan species (Balasov, 2009).

Orc6 is dispensable for DNA binding in budding yeast (Lee, 1997) but is required for origin recognition in Drosophila (Chesnikov, 2001; Balasov, 2007). Orc6 is tightly associated with core ORC subunits in yeast and Drosophila, and it is crucial for DNA replication in these species. In contrast, human Orc6 is loosely bound to the core complex (Vashee, 2001), and its role in DNA replication has not yet been completely established. This controversy has partially resulted from the use of different experimental systems that generally do not permit an organismal view of the DNA replication process under physiological conditions (Balasov, 2009).

The functional ORC complex begins to assemble on chromosomes during anaphase. It was found that during mitotic stages, Orc6 localization is remarkably similar to that of other ORC subunits, such as Drosophila Orc2 and Xenopus Orc1. All of these proteins were weakly associated with DNA at metaphase but present at the later stages of mitosis. This pattern of binding of Drosophila ORC to the chromatin depends on the cessation of mitotic cyclin activity (Baldinger, 2009). Most likely, at these stages functional 6-subunit ORC is deposited onto the replication origins in preparation for the next cell cycle (Balasov, 2009).

The analysis of a mutant phenotype for the Orc6 gene in Drosophila during the course of these studies confirmed, in a living animal, the important role of Orc6 in cell functions. Similarly to the other reported ORC mutants, vastly reduced BrdU labeling was observed in the proliferative neural tissue of larvae homozygous for the Orc6 mutation, consistent with a crucial role for ORC in the DNA replication. These data together with the increased occurrence of cells with a single centrosome indicate an early cell cycle delay, resulting in G1 arrest in Orc6-null mutant neuroblasts. The amount of cells arrested in G1 increased ~7-fold compared with the wild-type or heterozygous cells, suggesting that the inactivation of Orc6 leads to defects in pre-RC assembly. Some of the orc635 mutant cells, however, accumulated at a stage with many characteristic features of metaphase. Metaphase arrest with abnormally condensed chromosomes has also been observed in other ORC and replication protein mutants. It appears that in all of these cases, insufficient levels of replication proteins due to gradually depleted maternal deposits result in incomplete DNA replication and broken chromosomes that would lead to cell cycle arrest at the early stages of mitosis by a mechanism sensitive to chromosomal integrity (Balasov, 2009).

Recent studies on an Orc1 mutant suggested that ORC may be dispensable for the endoreplication in Drosophila (Park, 2008). Analysis of the Orc6-deletion mutant similarly revealed that despite DNA replication defects observed in Drosophila brain cells, the development and DNA synthesis in salivary glands were similar to the wild-type or heterozygous cells. Western blotting experiments did not reveal detectable Orc6 protein in salivary glands isolated from homozygous mutant animals. It is possible that as maternal supplies of Orc6 are depleted, the Western blotting analysis may not be sensitive enough to detect the residual Orc6 protein. Immunofluorescence experiments, however, consistently showed Orc6-specific bands throughout polytene chromosomes at the same stage. The number of the Orc6-positive bands was ~5- to 10-fold lower in polytene chromosomes derived from Orc6-deletion mutant cells than in chromosomes from control wild-type or heterozygous cells, but they were always detectable with many independent batches of affinity-purified rabbit polyclonal or mouse monoclonal antibody against Drosophila Orc6. Thus, it is not possible at this point to exclude the possibility that the residual maternal deposits of Orc6 protein are sufficient to support endoreplication in the salivary glands, augmented by a lower turnover of ORC in polyploid cells than in mitotically active cells (Balasov, 2009).

From earlier biochemical and cell culture studies of Orc6 activities, it was concluded that Orc6 protein in Drosophila consisted of 2 functional domains important for DNA binding and replication (Balasov, 2007), as well as for cytokinesis, through interaction with the septin protein Pnut (Chesnokov, 2003; Huijbregts, 2009). In the current study, the functions of Drosophila Orc6 were addressed in the living organism. Only the constructs containing a full-length Drosophila Orc6 gene, but not its C-terminal mutants, were able to rescue orc6- animals to viability, indicating that both domains of Orc6 are important for animal survival. C-terminal mutants of Orc6 were shown to be active in replication and DNA binding in vitro and in cell culture assays. Therefore, a priori at least partial rescue of DNA replication was expected in developing larval tissues expressing these mutants. Indeed, constructs containing Drosophila Orc6-220 deletion mutant and Orc6-WK228AA, and also human Orc6, were able to rescue DNA replication in brain neuroblasts of orc635 larvae. Cells arrested previously at G1 stage were now able to pass G1 arrest block and undergo a round of DNA replication. However, brains in orc635 animals containing these transgenes were still significantly smaller and undeveloped compared with the wild-type or heterozygous flies. Imaginal discs were also not detected, suggesting that normal cell cycle progression and tissue development were not restored. Consistent with this observation, a significant increase was found in neuroblasts arrested during mitosis, indicating that the N-terminal domain of Orc6 is not sufficient to rescue the mitotic function of the protein. It is also interesting to note that the Orc6-200 mutant, which was active in DNA binding and replication in vitro, failed to rescue DNA replication in the current in vivo study. This shorter Orc6-200 protein was also less tightly associated with core ORC1-5 subunits during reconstitution and purification assays, suggesting that this particular deletion may extend deeper into the replicative domain of the protein, resulting in observed replication defects in live animals (Balasov, 2009).

Orc6 is the least conserved ORC subunit; however, human Orc6 protein was able to rescue DNA replication in Drosophila larval brains lacking the endogenous Orc6. Orc6 in human cells is loosely bound to core ORC and also found to be dispensable for DNA binding and DNA replication on Orc2-depleted Xenopus egg extracts. Other studies have shown that Orc6 is important for DNA replication, because depletion of the protein from human cells results in DNA replication defects. In the current studies the expression of human Orc6 in Drosophila orc6- cells relieved an observed G1 arrest in orc635 mutants, resulting in a rescue of DNA replication. The ability of human Orc6 protein to support DNA replication in Drosophila cells suggests that the two proteins are homologous in replication function and also provides an opportunity for further molecular dissection of human Orc6 in vivo, by using Drosophila as a model system (Balasov, 2009).

Despite their ability to replicate DNA, the orc635 mutant cells carrying the Orc6-220, Orc6-WK228AA, and human Orc6 transgenes accumulated at the stage of mitosis with the significant increase in the number of mitotic cells, compared with orc6-null mutant. Many cells were arrested with metaphase-like figures with uncondensed and broken chromosomes, suggesting that DNA may be underreplicated in these mutants. Arrest at metaphase also suggests that C-terminal Orc6 mutants may be defective during ORC-dependent assembly of pre-RC at the final stages of mitosis. It is possible that the N terminus of Orc6 is necessary, but not wholly sufficient, to rescue all Orc6 functions in DNA replication. However, cells at different mitotic stages were found, suggesting that some cells escape metaphase arrest. Furthermore, the appearance of multinucleated and polyploid cells as well as cells with multiple centrosomes indicates that cell cycle progression through mitosis and cytokinesis is defective in cells carrying C-terminal mutations of Orc6 protein. The role of ORC subunits in coordinating DNA replication and centrosome copy number in human cells has been reported recently (Hemerly, 2009; Balasov, 2009 and references therein).

Overall, these data provide evidence in a living organism that DNA replication function of Orc6 is associated mainly with its N-terminal domain, whereas the C-terminal domain is necessary for the passage through the M phase. The whole Orc6 protein, however, is required for the survival of Drosophila. DNA replication function of Orc6 can also be rescued by human Orc6 protein, suggesting the conservation of replicative function among metazoan species (Balasov, 2009).

Drosophila Orc6 facilitates GTPase activity and filament formation of the septin complex

This study describes the analysis of the interaction of Orc6 with Pnut and whole Drosophila septin complex. Septin complex was purified from Drosophila embryos and also reconstituted from recombinant proteins. The interaction of Orc6 with the septin complex is dependent on the coiled-coil domain of Pnut. Furthermore, the binding of Orc6 to Pnut increases the intrinsic GTPase activity of the Drosophila septin complex, whereas in the absence of GTP it enhances septin complex filament formation. These results suggest an active role for Orc6 in septin complex function. Orc6 might be a part of a control mechanism directing the cytokinesis machinery during the final steps of mitosis (Huijbregts, 2009).

Both in Drosophila, a considerable pool of Orc6 is cytoplasmic, and the protein is either associated with or proximal to the plasma membrane and cleavage furrows of dividing cells. In Drosophila, Orc6 and Pnut colocalize in vivo at cell membranes and cleavage furrows of dividing cells, and during cellularization in Drosophila early embryos (Chesnokov, 2001). The C-terminal domain of Orc6 is necessary for this colocalization with Pnut. Moreover, Orc6 RNAi results in cytokinesis defects in Drosophila tissue culture cells (Chesnokov, 2003), whereas Pnut RNAi disrupts the localization of Orc6 to the plasma membrane (Huijbregts, 2009).

Analysis of the cells treated with Pnut dsRNA revealed an elevated number of binucleated cells (5- to 30-fold increase, depending on the experiment). This is in contrast with previously reported data in which no elevated numbers of binucleated cells were detected in cultures treated with Pnut dsRNA. Differences in culture conditions, amount of dsRNA used, as well as cell preparation protocols for analysis might have contributed to the discrepancies between the two studies (Huijbregts, 2009).

Deletion of part of the predicted coiled-coil domain of Pnut impaired its ability to form a complex with both Sep1 and Sep2 together, but it was still able to interact with Sep1. A previous study proposed that different septin complexes may exist within Drosophila. The current data suggests that Pnut and Sep1 might form a precomplex that joins with Sep2 to form the complete septin complex. The interaction of Orc6 with Pnut is also disrupted in C-terminal deletion mutants of Pnut, suggesting that the coiled-coil domain of Pnut is important for both binding with Orc6 and for the formation of the septin complex. However, leucine to alanine substitutions within the coiled-coil domain of Pnut prevent Orc6 binding but do not inhibit complex assembly, indicating that these protein interactions are based on different structural moieties within the C terminus. Furthermore, direct interaction studies revealed that the coil-coil domain of Pnut is not sufficient for the interaction of this septin with Orc6 and that other structural features may also be important for the interaction between the two proteins (Huijbregts, 2009).

To study the effect of Pnut mutations in vivo in Drosophila tissue culture cells, various expression systems were used, including heat shock and metallothionein promoters. In all cases, expression of GFP-Pnut in L2 cells resulted in rod- and spiral-like structures present throughout the cytosol. Expression of either N-terminal or C-terminal GFP fusions to Pnut, or a FLAG-Pnut protein also resulted in the same aberrant structures, compromising the in vivo analysis of Pnut mutants. However, when under control of the native Pnut promoter, proteins could be expressed in L2 cells at lower levels. This figure further shows that the coiled-coil domain of Pnut, which is important for Drosophila septin complex assembly, also is essential for the in vivo localization of Pnut, because FLAG-Pnut(1-427), lacking the coiled-coil domain, had the tendency to accumulate into crescent shaped aggregates. The FLAG-tagged triple leucine mutants of Pnut exhibited mainly diffuse cytoplasmic staining when expressed in L2 cells, although some plasma membrane staining was observed. It is possible that due to the mutations in the coiled-coil domain these Pnut mutants do not interact properly with other proteins (as shown for Orc6), resulting in a release from the plasma membrane at specific cell stages (Huijbregts, 2009).

Native septin complex as well as reconstituted septin complexes exhibit the characteristic properties of filament formation and GTPase activity, indicating that they are functional complexes. Because insect cell lines are closely related to Drosophila, baculovirus-derived reconstituted septin complex was used for further biochemical studies (Huijbregts, 2009).

The human SEPT2-SEPT6-SEPT7 complex can be formed from recombinant proteins all lacking their predicted coiled-coil domains, suggesting that their C termini are dispensable for complex formation (Sirajuddin, 2007). Structural analysis of crystals of the human septin complex revealed that the filaments consist of an assembly of GTP binding domains. However, the coiled-coil domains of SEPT6 and SEPT7 do interact directly with each other, suggesting that although not required for the human septin complex, coiled-coils may further stabilize filament formation (Sirajuddin, 2007). The GTP binding domains of human septin proteins can also interact with coiled-coil structures within the multiple subunit complex. This might also occur with the Drosophila septins when they assemble into complex. However, the interaction of Orc6 with the septin complex seems strongly dependent on the coiled-coil domain of Pnut (Huijbregts, 2009).

One possible role of the interaction of Orc6 with the septin complex could be the regulation of the GTPase activity of the complex during cytokinesis. Orc6 reproducibly increased the GDP-to-GTP ratio of bound nucleotide of the whole septin complex, but no significant increase of total nucleotide bound to complex was detected. It has been hypothesized that GTP hydrolysis might promote disassembly of the septin complex. However, purified recombinant septin complex was retrieved intact with bound Orc6 after 2-h incubation in the presence of GTP, although potentially the disassembly of a small amount of complex might have occurred. It was observed that many larger filaments present in concentrated recombinant septin complex samples were not detected under GTPase assay conditions, most likely due to the dilution of concentrated sample. No differences in filament size were observed for septin complexes incubated either in the presence or absence of GTP. However, due to limitations of the EM setup, subtle changes in small filament size could not be detected (Huijbregts, 2009).

Although GTP hydrolysis by septin complex was accelerated by Orc6 binding (because the presence of a nonbinding mutant of Orc6 had no additional affect on hydrolysis), no significant changes could be detected in turnover rate. The higher turnover rates reported for individual Xenopus, mouse, and human recombinant septin proteins do not exclude a regulatory function for these subunits in vivo when not assembled in complex. A structural rather than regulatory role for septin complex-bound GTP and GDP was proposed from the results obtained with yeast septins. No turnover of yeast septin-bound GTP and GDP could be detected during a cell cycle in vivo. Furthermore, in vitro experiments revealed that GTP hydrolysis of yeast septin complex was limited by its slow binding or exchange activity, similar to the properties described initially for the Drosophila septin complex. The role of Orc6 in GTP hydrolysis and filament disassembly of septin complex also suggests that in the case of Drosophila the guanine nucleotides bound to septins may contribute to the structural properties of the complex. Additionally, the importance of the GTP binding domains for the assembly of the human septin complex and potentially filament formation (Sirajuddin, 2007) also indicates a role for guanine nucleotide in septin complex structure (Huijbregts, 2009).

The addition of Orc6 to septin complex in the absence of GTP, in contrast, greatly induced filament formation, whereas in the presence of GTP the effect was not observed. This indicates that Orc6 exhibits two opposite effects in its interactions with the septin complex. Based on the sequence homologies between human or Drosophila septins, hexamers can be depicted as a linear protein similar to the crystal structure of the human septin complex (Sirajuddin, 2007) with Pnut at either end of the complex (see Model for the interaction of Orc6 with the septin complex). In the absence (or low concentration) of GTP Orc6 binding to Pnut enhances linear filament assembly, potentially due to conformational changes in either Pnut or other septin subunits. Orc6 stabilizes the formation of the filaments by protein-protein interactions. It is interesting to note that purified recombinant Orc6 protein behaves as a dimer in biochemical assays. In the presence of GTP, Orc6 increases the GTPase activity of the septin complex, at the same time resulting in filament disassembly. Increased GTPase activity may lead to conformational changes in the septin complex, causing disassembly of septin filaments. These results suggest that Orc6 may regulate either assembly or disassembly of septin filaments. Whether Orc6 actively induces filament disassembly in the presence of GTP or this process is a result of the increased GTPase activity of the septin complex remains to be investigated (Huijbregts, 2009).

The septins are important for cytokinesis but molecular mechanisms of their functions in this process are not completely understood. These data on interactions between Drosophila Orc6 and the septin complex reveal some new aspects for these proteins. Orc6 has an effect on both GTPase activity and filament formation of the septin complex, suggesting that Orc6 might have a direct role in septin complex functions during the last stage of mitosis (Huijbregts, 2009).

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. The smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. The core replication domain of the Orc6 protein, which does not include the C-terminal domain, is defined in this study. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. Mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. These studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins (Balasov, 2007).

The role of Orc6 in Drosophila is particularly interesting, since in budding yeast this subunit is dispensable for DNA binding. Drosophila ORC is also different from Xenopus and human ORC in the avidity of Orc6 association, suggesting that Orc6 may be involved in differential regulation of ORC in these organisms. Therefore, given the crucial and highly conserved role of ORC during replication initiation, it is very interesting that the DNA binding ability of ORC appears to be mediated by different subunits (or their combinations) in different species. Previous work showed that Drosophila Orc6 is important for ORC-dependent DNA binding and DNA replication. Orc6 includes two distinct functional domains. The long N-terminal region is important for DNA replication, whereas the smaller 57-amino-acid C-terminal domain interacts with the septin protein Peanut and is involved in cytokinesis. These findings were confirmed by biochemical and cell-based genetic assays. It was found that ORC(1-5) protein lacking the Orc6 subunit cannot bind DNA efficiently and does not support ORC-dependent DNA replication in Drosophila extracts, suggesting that Orc6 is strongly involved in both of these activities (Balasov, 2007).

This study showed that Orc6 by itself binds DNA with a preference for poly(dA) sequences. Synthetic DNAs with poly(dA) tracts competed successfully for binding of Orc6 to genomic DNA fragments containing origins of replication in Drosophila. Poly(dA) · poly(dT) was ~10 to 100-fold better than any other synthetic DNA tested. Sequence analysis of the ACE3 and ori-ß fragments of Drosophila origins shows that these sequences are enriched with poly(dA) and poly(dT) tracts compared to other fragments derived from the chorion gene amplification locus. In contrast, the average AT content of these fragments does not differ significantly throughout the locus. This explains preferential binding of Orc6 and reconstituted Drosophila ORC to ACE3 and ori-ß fragments. Austin (1999) has shown that ORC preferentially binds to the ACE3 fragment both in vitro and in vivo but did not quantitatively address the issue of ORC-DNA binding specificity. ORC associates with AT-rich Sciara coprophila origin II/9A but not with flanking fragments in vivo, and reconstituted Drosophila ORC binds the same sequence in vitro. Remus (2004) used EMSAs to test quantitatively the relative affinity of Drosophila ORC to various fragments derived from the third chromosome chorion gene cluster that included both ACE3 and ori-ß regions. It was concluded that the ORC DNA binding to the 'nonspecific' sequences was up to sixfold lower than the 'specific' (ACE3 and ori-ß) fragments and that the topological state of the DNA significantly influences the affinity of ORC to DNA. Genome-wide analysis demonstrated that ORC in Drosophila localizes to AT-rich chromosomal sites, many of which coincide with early replication origins. Moreover, ORC was excluded from sequences with low AT content in these experiments, suggesting that increased AT content is necessary for ORC association. In agreement with these data, this study found that Orc6 overexpressed in Drosophila salivary glands localizes at interband regions in polytene chromosomes. Interband regions are extremely AT rich with a high concentration of poly(dA) stretches. The same regions replicated early during amplification of these giant chromosomes (Balasov, 2007 and references therein).

Thus, DNA-binding properties of the Orc6 protein and reconstituted Drosophila ORC might explain the connection between origins of DNA replication and poly(dA) blocks in vivo. The observed preference of Orc6 for poly(dA) tracts and interband regions of polytene chromosomes might be explained in part by the chromatin structure associated with these sequences. Interestingly, poly(dA) tracts form a rigid structure that is hard to deform and do not wrap easily around nucleosome cores. This feature of poly(dA) sequences might provide the basis for the assembly of a less condensed chromosome structure with a low package ratio, like interbands, making it more open and accessible to proteins like Orc6 and ORC (Balasov, 2007).

The preferential binding of Drosophila Orc6 and ORC to synthetic AT-rich DNA is also seen in the fission yeast S. pombe ORC. DNA binding of the S. pombe ORC to AT-rich DNA is mediated by a unique N-terminal domain in the Orc4 subunit, which contains nine AT-hook motifs known to make minor groove contacts. S. pombe ORC binds more selectively than Drosophila ORC and human ORC to AT-rich DNA, and AT tracts have been shown to be important for the function of some origins in S. pombe, whereas no such requirement has been demonstrated for metazoan origins. It is particularly interesting that S. pombe origins can be substituted by clustered poly(dA) stretches without noticeably affecting origin functions (Balasov, 2007).

The helical structure of A-tract DNA is often referred to as the B* form. Characteristic features of the B*-form helix include an unusually narrow minor groove and a high base propeller twist. The most peculiar and intensely studied feature of A-tract sequences is their propensity to cause helical axis bending when incorporated into otherwise non-A-tract DNA, which may promote DNA-protein interactions. A similar structure of a minor groove is found in poly(dI) · poly(dC) polymers, which might explain the ability of poly(dI) · poly(dC) to compete successfully for Orc6 in EMSA experiments. However, Orc6 does not contain canonical AT-hook consensus sequences, so any minor groove interactions must be mediated by other structural motifs. To identify these motifs molecular modeling was used together with sequence comparison of metazoan Orc6 homologues. Aside from the hypothetical nature of this modeling, several points are emphasized here. First, the break in a predicted TFIIB homology domain occurs at amino acid 203 of the Drosophila Orc6 protein sequence, in good agreement with biochemical and cell-based genetic assays. The C-terminal domain, which has been shown to be important for cytokinesis, does not fit into this fold. Second, the sequence comparison between Orc6 metazoan homologues revealed conserved amino acids that form a putative helix-turn-helix motif in the model. A corresponding structural motif is important for DNA recognition by TFIIB. Third, point mutations of two amino acids within this structural motif abolished DNA binding activities of Orc6 and, even more importantly, severely compromise DNA binding and DNA replication activities of reconstituted Drosophila ORC containing mutant Orc6 subunits. Moreover, mutant Orc6 proteins had an inhibitory effect on DNA replication in vivo, consistent with the requirement of Orc6 for ORC-dependent DNA binding and replication (Balasov, 2007).

Of all metazoan ORCs, only Drosophila, Xenopus, and human proteins have been purified and biochemically characterized in detail. Drosophila and human ORCs display similar DNA binding activities. Both Drosophila and human ORCs can bind DNA in the absence of ATP, although DNA binding is stimulated two- to fivefold by ATP. Although the Drosophila ORC, like the human ORC, can bind nonspecifically to many different DNA sequences in vitro, both proteins exhibit a preference for AT-rich sites. Drosophila ORC localizes preferentially to AT-rich ACE3 and ori-ß sequences in vivo and binds the same fragments in vitro. Recombinant reconstituted human ORC shows a preference for poly(dA) sequences in vitro. It would be interesting to investigate if the human Orc6 protein, similar to the Drosophila homologue, displays an affinity for poly(dA) sequences (Balasov, 2007).

All experimental data available to this date indicate that Orc6 is essential for ORC-dependent DNA binding and DNA replication in Drosophila. In Xenopus and human systems, published data suggest that Orc6 may not be important for these activities. This apparent inconsistency may reflect the difference in affinity of Orc6 for core ORC(1-5) complex in distant metazoan species. Drosophila ORC purifies as a tight six-subunit complex, even though a free pool of the Orc6 subunit is detected during purification from Drosophila egg extracts. In contrast, Xenopus and human Orc6 subunits are consistently underrepresented compared with the other subunits in preparations of Xenopus and human ORC, either recombinant or purified from extracts. It appears that in Xenopus and humans, Orc6 is less tightly associated with the core complex than other subunits and can be purified separately. During in vitro replication reactions in Xenopus egg extracts, recombinant human ORC was able to initiate DNA replication from essentially any DNA sequence. Human ORC(1-5) is able to restore DNA replication in Xenopus extracts depleted for ORC using antibody against the Orc2 subunit. ORC(1-5) was shown to be sufficient for licensing of replication origins in Xenopus. As Orc6 is less tightly associated with ORC in human and Xenopus, it is possible that this subunit is not completely removed from the extract when antibodies, raised against other ORC subunits, are used for immunodepletion. In Drosophila, ORC(1-5) is unable to support DNA replication when extracts are immunodepleted of both ORC(1-5) and Orc6. The presence of a free pool of Orc6 in the early Drosophila egg extract, if not depleted, restores a functional six-subunit ORC that is active in both chromatin binding and DNA replication. It would be interesting to see whether immunodepletion of Orc6 from Xenopus extract has an effect on replication and licensing activities of human and/or Xenopus ORC(1-5). One recent result suggests that this indeed might be a case. The addition of either the Xenopus or human Orc6 subunit together with recombinant human ORC(1-5) to Xenopus extracts immunodepleted of all six subunits of ORC significantly increased the ability of human ORC to replicate DNA in Xenopus extracts (J. Blow and M. Gossen, personal communication to Balasov, 2007), providing evidence that Orc6 might act as an assembly and/or activation factor for ORC(1-5) (Balasov, 2007 and references therein).

In conclusion, these data strongly indicate that Orc6 in Drosophila is a DNA binding subunit of ORC, which is necessary for ORC replicative function. Orc6 is an integral part of the entire complex and functions in targeting ORC to the origins of DNA replication. Orc6 binds DNA directly, but it does not recognize a specific sequence. Rather it has an affinity for structural and topological features associated with poly(dA) stretches such as a minor groove structure. The importance of the topological state of DNA for the entire Drosophila ORC DNA binding activity has been shown recently (Remus, 2004). In vivo Orc6 associates with AT-rich, early replicating, interband regions. Genome-wide analysis of the entire ORC distribution in Drosophila revealed its preferential localization to AT-rich transcriptionally active chromosomal sites, many of which coincide with early replication origins. According to this model the binding of Drosophila ORC to the origin DNA is mediated by the Orc6 subunit, which works as an anchor and targets ORC to the origins of DNA replication. This initial binding step is followed by ATP-dependent binding of the entire ORC. Interestingly, human and Xenopus Orc6 proteins are less tightly associated with core ORC subunits and may act, according to this model, as an assembly factor for ORC at the origins of DNA replication. In this case the Orc6 protein marks the origins and helps target the core ORC(1-5) complex to the DNA. As a result, six-subunit ORC is assembled at the origin at the end of mitosis and activated to ensure correct and timely origin licensing. This model may serve as a unifying mechanism for the initial stages of ori recognition in all metazoan species (Balasov, 2007).

A cytokinetic function of Drosophila ORC6 protein resides in a domain distinct from its replication activity

Coordination between separate pathways may be facilitated by the requirements for common protein factors, a finding congruent with the link between proteins regulating DNA replication with other important cellular processes. This study found that the smallest of Drosophila origin recognition complex subunits, Orc6, was found in embryos and cell culture localized to the cell membrane and cleavage furrow during cell division as well as in the nucleus. A two-hybrid screen revealed that Orc6 interacts with the Drosophila Peanut (Pnut), a member of the septin family of proteins important for cell division. This interaction, mediated by a distinct C-terminal domain of Orc6, was substantiated in Drosophila cells by coimmunoprecipitation from extracts and cytological methods. Silencing of Orc6 expression with double-stranded RNA resulted in a formation of multinucleated cells and also reduced DNA replication. Deletion of the C-terminal Orc6-peanut interaction domain and subsequent overexpression of the Orc6 mutant protein resulted in the formation of multinucleated cells that had replicated DNA. This mutant protein does not localize to the membrane or cleavage furrows. These results suggest that Orc6 has evolved a domain critical mainly for cytokinesis (Chesnokov, 2003).

Septins are polymerizing proteins with a common GTPase activity and were first discovered in S. cerevisiae but now seem to be ubiquitous in fungi and animals. Pnut is one of the five Drosophila septins identified to the date. Genetic data showed that the proteins play diverse roles in organization of the cell cortex and in cytokinesis. At the molecular level, the role of GTP binding and hydrolysis by septins is unclear and is probably not required for filament formation. Thus, formation of filaments at a cleavage furrow or in the cytoplasm during interphase may be an activity of septins independent of other roles for the proteins, perhaps in intracellular signaling, because they seem to be more homologous to the ras superfamily members than to other GTPases. In many cell types examined to date, the septins form rings at the site of the cleavage furrow and septin mutants in S. cerevisiae and Drosophila are defective in cytokinesis. In Drosophila, Pnut is an essential protein. In pnut mutants, cells of the imaginal disk tissues fail to proliferate and instead develop clusters of large multinucleated cells, consistent with an important role in cytokinesis. In S. cerevisiae, septins are required for proper localization of bud site-selection markers Bud3p and Bud4p and of the subunits of the chitin synthase III complex. These and other data led to the hypothesis that the septins function as a scaffold on which other proteins assemble along the cytoplasmic side of the cleavage furrow. How septins themselves localize is unknown; adaptor proteins likely recruit them to specific targets (Chesnokov, 2003 and references therein).

Whole-cell extracts from Drosophila embryos (0-12 h of development) and L2 tissue culture cells were subjected to immunoprecipitation with polyclonal antibody raised against the Drosophila Orc6 subunit. Coprecipitated material was analyzed by Western blotting for the presence of Pnut protein. Polyclonal anti-Orc6 serum coimmunoprecipitates Pnut together with Orc6 protein from both Drosophila embryonic and L2 tissue culture extracts. No Pnut signal was detected in control reactions with polyclonal antibody against the Drosophila Orc2 subunit. The negative results with the Orc2 sera imply that it is the pool of Orc6 unassociated with the other ORC proteins that interacts with Pnut (Chesnokov, 2003).

Although Pnut could be coimmunoprecipitated with anti-Orc6 antibodies, the reciprocal experiment showed that only a very minor fraction of Orc6 was precipitated by monoclonal antibodies raised against Pnut protein. This result was perhaps due to epitope masking in the Orc6-Pnut complex, coupled with the fact that this particular Pnut monoclonal antibody worked best in immunostaining and rather poorly in immunoprecipitations (Chesnokov, 2003).

Both Drosophila embryos at different stages of development and L2 cells were used to determine whether Orc6 and Pnut proteins colocalize. At the stage of cellularization that occurs after the 13th nuclear division, the Pnut signal became apparent at the advancing membrane front and especially at the cytoplasmic connections between cells and yolk. These yolk plugs maintain actin, myosin, anillin, and the septins in a surrounding ring. Orc6 is indeed found with Pnut at these locations. Later, as cell membranes grow and eventually reach a full depth, Orc6 is also localized at the membrane locations together with Pnut protein. Orc6 also colocalizes with Pnut in 3.5- to 4-h-old embryos (stages 8 and 9). At these stages, shortly after gastrulation, cells enter cell cycle 15 when neuroblasts delaminate from the ectoderm. Both Drosophila Orc6 and Pnut proteins are found at the cleavage furrows between dividing cells in the presumptive neurogenic ectoderm (Chesnokov, 2003).

Double staining of Drosophila L2 tissue culture cells with Orc6 and Pnut antibodies also showed a colocalization in dividing cells. In addition to its anticipated nuclear localization, endogenous Orc6 is localized to the cell membranes together with Pnut protein. Moreover, in mitotic cells Orc6 and Pnut colocalize at the cleavage furrow of the dividing cells. Transient ectopic expression of the GFP-Orc6 fusion protein again showed distinct nuclear and membrane localization of the protein. Similar GFP fusions with ORC1 and two genes elicited only nuclear signals, and cytology with ORC2 antibodies detected only a nuclear stain in embryos (Chesnokov, 2003).

Because pnut mutations in Drosophila result in a cytokinesis defect, the role of Orc6 in this process was explored by depletion with RNAi. After transfection of the appropriate double-stranded RNA into an asynchronous population of Drosophila cells, immunoblot analysis of treated L2 cells revealed that the level of Orc6 protein was greatly reduced by 24 h and almost completely lost by 72 h. The level of Orc2 protein was not significantly decreased in cells treated with Orc6 dsRNA, and the cells transfected with luciferase dsRNA as a control showed normal levels of Orc2 and Orc6 proteins during the time course of this experiment. Immunostaining of transfected cells with anti-Orc6 antibody showed a concomitant disappearance of the Orc6 signal. DNA replication in cells treated with Orc6 dsRNA also decreased over time. BrdUrd incorporation was detected in 70%-80% of these cells after the first 24 h of incubation, similar to what was observed with untreated cells. However, the fraction of cells incorporating the precursor nucleotide dropped continuously (40%-50% at 48 h and 10%-15% at 72 h). The most striking phenotype observed after the transfection of L2 cells with Orc6 dsRNA was the rapid appearance of binucleated cells. The number of multinucleated cells observed increased from a background of <0.2% in the population to 5% after the first 24 h and reached ~30% after 72 h of transfection. Prolonged periods of the Orc6 depletion by RNAi resulted in a decrease of cell proliferation and increased cell death. This multinucleated phenotype was not observed in vivo for lethal mutations of ORC subunits 2, 3, or 5. Moreover, dsRNA for ORC2, although effective for decreasing BrdUrd incorporation, showed no efficacy for elevating the number of binucleated cells above the background. However, such defects were observed in RNAi-based studies of Drosophila passenger proteins INCENP and Aurora B. From these data, it is speculated that Orc6 participates in some aspects of the cell division cycle that influences cytokinesis. Furthermore, the kinetics of cytokinesis defects suggest that the cytokinetic function is more sensitive to small changes in the Orc6 pool than is DNA replication (Chesnokov, 2003).

The C-terminal 25 aa of Drosophila Orc6 contains a leucine-rich region that may mediate protein-protein interactions through an amphipathic helix. A similar motif, thought to be important for protein-protein interaction, is also found in the Pnut protein and is present in most of the septins described to date. The notion that this region of Orc6 is important for Pnut interaction was tested with a series of Orc6 C-terminal deletion mutants. Purified WT and mutant proteins expressed in E. coli as His-tagged fusions were tested for their ability to precipitate Pnut protein from the Drosophila cell culture extracts. Proteins were precipitated by using cobalt beads (Qiagen) that can bind selectively His-tagged proteins. Precipitated material was analyzed by employing a Western immunoblotting assay and the anti-Pnut antibody for detection. In contrast to the WT Orc6 His-tagged protein, Orc6 mutant protein lacking the terminal 57 aa (Orc6-200) failed to precipitate Pnut from the Drosophila extracts. Orc6 protein truncated at the terminal 33 aa (Orc6-224) was able to precipitate Pnut protein but less efficiently than the intact protein. Thus, the terminal leucine-rich section of Orc6 is important for Pnut interaction but not likely the sole mediator. Equivalent results were obtained with Drosophila embryonic extracts (Chesnokov, 2003).

It was asked whether these deletion mutants might have dominant-negative effects if expressed in cultured cells. Overexpression of WT Orc6 protein does not produce any noticeable effect on either cell morphology or the ability of the cells to replicate DNA in side-by-side comparison to nontransfected cells. However, the overexpression of C-terminal Orc6 mutants resulted in an elevated number of cells with multiple nuclei (5%-7% of transfected cells for Orc6-224 and up to 30% for Orc6-200). Overexpression of the Orc6-200 allele also results in a loss of membrane localization. Cells carrying GFP-Orc6-224 or GFP-Orc6-200 were able to incorporate BrdUrd during a 20-h labeling period at the level of nontransfected cells or cells transfected with GFP-Orc6 WT, as judged by intensity of staining and the fraction of cells with signal. Further, by using a recombinant ORC system, the ORC6-200 allele, when expressed with the other subunits, incorporates effectively into the complex and thus can compete with the WT gene for complex formation when it is overexpressed. This result suggests that the core replication domain of Orc6 is not affected by these mutations, although the cytokinetic function as measured by the presence of binucleated cells is lost. The antimorphic nature of alleles such as GFP-ORC6-200 indicates that the replication domain may also contain critical functions for cytokinesis, but that in the absence of the C terminus the defective protein interferes with the process. For example, the ORC6-200 may bind and sequester a protein important for releasing ORC6 from chromatin to transport the protein to the membrane locations. Overexpression of Orc6-163 mutant protein, in contrast, results in large cells that do not incorporate BrdUrd. Moreover, transfection of L2 cells with Orc6-163 apparently has a toxic effect on cells and results in decreased proliferation and increased cell death (Chesnokov, 2003).

A reasonable extrapolation from these data is that a C-terminal domain of Orc6, perhaps beginning around amino acid 200 and progressing from there toward the C terminus, defines a Pnut interaction domain and a membrane-proximal localization function critical for cytokinesis. To probe the domain organization of Orc6 in silico, a web-based method for protein fold prediction was used that employs 1D and 3D sequence profiles coupled with secondary structure and solvation potential information. With this program, Drosophila and human Orc6 sequences were compared with known protein structures. Unexpectedly, it was found that the predicted Orc6 structure over much of its length was homologous to the structure of the human TFIIB transcription factor bound to the DNA in a complex with TBP. The E value, an inverse measure of the program's reliability for this alignment, was commensurate with a certainty of >99.9% for the human Orc6 and 99.2% for the Drosophila Orc6 homologue. Two points aside from the hypothetical nature of this modeling are emphasized here. (1) The break in the predicted TFIIB homology domain is at amino acid 203, and the C-terminal amino acids of Orc6 do not fit into this fold, in rather good agreement with biochemical and cell-based genetic assays. Deletion alleles map an approximate break in functional activities to this region. (2) A recent report also presented data consistent with a cleavage furrow localization of human Orc6 and function in cytokinesis. Ablation of human ORC6 expression in cultured cells via the RNAi method also leads to a rapid appearance of binucleated cells and a decrease in DNA replication. The domain structure homology predictions are noted as even higher for the human protein (Chesnokov, 2003).

It was asked whether the C-terminal region of Orc6 was both necessary and sufficient for Pnut interaction as anticipated from the hypothesis that this region defines a discrete domain of the protein. A peptide corresponding to the last 71 aa of Drosophila Orc6 protein was synthesized, linked by a spacer to biotin, and used to investigate this point. The biotin-labeled peptide was incubated with Drosophila embryonic whole-cell extract and then reisolated by using paramagnetic beads coupled with streptavidin (Promega). The bound material was subjected to SDS/PAGE and analyzed by Western blotting for the presence of Drosophila Pnut protein. The Orc6 C-terminal peptide was able to precipitate Pnut from the extract with a concentration as low as 5-10 micromol. It is concluded that this C-terminal region is a distinct domain of the protein. The putative structural homology between ORC6 and TFIIB can be tested, for example, by physical methods, and if borne out it would bring forth the notion that certain proteins involved in the initiation of replication coevolved with proteins important for transcription. In this context, it is intriguing that archael organisms have a single gene encoding an Orc1 family member and a TFB (TFIIB homolog); perhaps the respective encoded proteins interact (Chesnokov, 2003).

The primary question raised by these findings might be posed as follows: Does the role of Orc6 in cytokinesis actually link the regulation of DNA replication to this late step in cell division? A priori, it might be envisioned that the first steps toward building a prereplication complex in early G1 or late telophase might be tied to the successful completion of cytokinesis. Orc6 molecules at the cleavage furrow might participate in some event during cytokinesis and then after execution shuttle to chromosomes perhaps with other proteins. This shuttling might make dependent the completion of a cytokinetic function to the start of a new round of replication. This model posits a late step in cytokinesis for ORC6 that might couple the cytokinetic and DNA replication pathways. Alternatively, Orc6 may participate in some early role in cytokinesis assisting in a targeting function for septins in metazoans, thus potentially linking assembly of such septin rings at the cleavage furrow to the completion of DNA replication (Chesnokov, 2003).

Functional analysis of mutant and wild-type Drosophila origin recognition complex

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 results in complexes that could be purified to homogeneity through four chromatographic steps, and the mutant complexes assemble and exhibite 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 support 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 ORC 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 hydrolyze 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 was 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 origin 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).


REFERENCES

Search PubMed for articles about Drosophila Orc6

Austin, R. J., Orr-Weaver, T. L. and Bell, S. P. (1999). Drosophila ORC specifically binds to ACE3, an origin of DNA replication control element. Genes Dev. 13: 2639-2649. PubMed ID: 10541550

Balasov, M., Huijbregts, R. P. and Chesnokov, I. (2007). Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster. Mol. Cell. Biol, 27: 3143-3153. PubMed ID: 17283052

Balasov, M., Huijbregts, R. P. and Chesnokov, I. (2009). Functional analysis of an Orc6 mutant in Drosophila. Proc. Natl. Acad. Sci. 106(26): 10672-7. PubMed ID: 19541634

Baldinger, T. and Gossen, M. (2009). Binding of Drosophila ORC proteins to anaphase chromosomes requires cessation of mitotic cyclin-dependent kinase activity. Mol. Cell. Biol. 29: 140-149. PubMed ID: 18955499

Chen, S., de Vries, M. A. and Bell, S. P. (2007). Orc6 is required for dynamic recruitment of Cdt1 during repeated Mcm2-7 loading. Genes Dev. 21: 2897-2907. PubMed ID: 18006685

Chesnokov, I., Remus, D. and Botchan, M. (2001). Functional analysis of mutant and wild-type Drosophila origin recognition complex. Proc. Natl. Acad. Sci. 98(21): 11997-2002. PubMed ID: 11593009

Chesnokov, I. N., Chesnokova, O. N. and Botchan, M. (2003). A cytokinetic function of Drosophila ORC6 protein resides in a domain distinct from its replication activity. Proc. Natl. Acad. Sci. 100(16): 9150-5. PubMed ID: 12878722

Hemerly, A.S., Prasanth, S. G., Siddiqui, K., and Stillman. B. (2009). Orc1 controls centriole and centrosome copy number in human cells. Science 323: 789-793. PubMed ID: 19197067

Huijbregts, R. P., et al. (2009). Drosophila Orc6 facilitates GTPase activity and filament formation of the septin complex. Mol. Biol. Cell. 20(1): 270-81. PubMed ID: 18987337

Lee, D. G. and Bell, S. P. (1997). Architecture of the yeast origin recognition complex bound to origins of DNA replication. Mol. Cell. Biol. 17: 7159-7168. PubMed ID: 9372948

Li, J. J. and Herskowitz, I. (1993). Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science 262: 1870-1874. PubMed ID: 8266075

Newlon, C. S. (1993). Two jobs for the origin replication complex. Science 262: 1830-1831. PubMed ID: 8266070

Park, S. Y. and Asano, M. (2008). The origin recognition complex is dispensable for endoreplication in Drosophila. Proc. Natl. Acad. Sci. 105: 12343-12348. PubMed ID: 18711130

Prasanth, S. G., Prasanth, K. V. and Stillman. B. (2002). Orc6 involved in DNA replication, chromosome segregation, and cytokinesis. Science 297: 1026-1031. PubMed ID: 12169736

Remus, D., Beall, E. L. and Botchan, M. R. (2004). DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding. EMBO J. 23: 897-907. PubMed ID: 14765124

Semple, J. W., et al. (2006) An essential role for Orc6 in DNA replication through maintenance of pre-replicative complexes. EMBO J. 25: 5150-5158. PubMed ID: 17053779

Sirajuddin, M., Farkasovsky, M., Hauer, F., Kuhlmann, D., Macara, I. G., Weyand, M., Stark, H., and Wittinghofer, A. (2007). Structural insight into filament formation by mammalian septins. Nature 449: 311-315. PubMed ID: 17637674

Vashee, S., Simancek, P., Challberg, M. D. and Kelly, T. J. (2001) Assembly of the human origin recognition complex. J. Biol. Chem. 276: 26666-26673. PubMed ID: 11323433


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