Origin recognition complex subunit 2
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).
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).
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).
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).
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).
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).
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