Origin recognition complex subunit 1


Cloning and functional characterization of yeast ORC1

The origin recognition complex (ORC), a multisubunit protein identified in Saccharomyces cerevisiae, binds to chromosomal replicators and is required for the initiation of cellular DNA replication. Complementary DNAs (cDNAs) encoding proteins related to the two largest subunits of ORC were cloned from various eukaryotes. The cDNAs encoding proteins related to S. cerevisiae Orc1p were cloned from the budding yeast Kluyveromyces lactis, the fission yeast Schizosaccharomyces pombe, and human cells. These proteins show similarity to regulators of the S and M phases of the cell cycle. Genetic analysis of orc1+ from S. pombe reveals that it is essential for cell viability. The cDNAs encoding proteins related to S. cerevisiae Orc2p were cloned from Arabidopsis thaliana, Caenorhabditis elegans, and human cells. The human ORC-related proteins interact in vivo to form a complex. These studies suggest that ORC subunits are conserved and that the role of ORC is a general feature of eukaryotic DNA replication (Gavin, 1995).

The origin recognition complex (ORC) is a six protein assembly that binds S. cerevisiae origins of replication and directs DNA replication throughout the genome and transcriptional silencing at the yeast mating-type loci. Genes encoding the 120 kDa (ORC1), 62 kDa (ORC3), and 56 kDa (ORC4) subunits of ORC have been cloned and the complete complex has been reconstructed after expression of all six subunits in insect cells. Orc1p is related to Cdc6p and Cdc18p, which regulate DNA replication and mitosis, and to Sir3p, a regulator of transcriptional silencing. The N-terminal region of Orc1p is highly related to Sir3p, and studies of Orc1p/Sir3p chimeric proteins indicate that this domain is dedicated to the transcriptional silencing function of ORC (Bell, 1995).

cdc18+ of Schizosaccharomyces pombe is a periodically expressed gene that is required for entry into S phase and for the coordination of S phase with mitosis. cdc18+ is related to the Saccharomyces cerevisiae gene CDC6, which has also been implicated in the control of DNA replication. A new Sch. pombe gene, orp1+, has been identified that encodes an 80-kDa protein with amino acid sequence motifs conserved in the Cdc18 and Cdc6 proteins. Genetic analysis indicates that orp1+ is essential for viability. Germinating spores lacking the orp1+ gene are capable of undergoing one or more rounds of DNA replication but fail to progress further, arresting as long cells with a variety of deranged nuclear structures. Unlike cdc18+, orp1+ is expressed constitutively during the cell cycle. cdc18+, CDC6, and orp1+ belong to a family of related genes that also includes the gene ORC1, which encodes a subunit of the origin recognition complex (ORC) of S. cerevisiae. The products of this gene family share a 250-amino acid domain that is highly conserved in evolution and contains several characteristic motifs, including a consensus purine nucleotide-binding motif. Among the members of this gene family, orp1+ is most closely related to S. cerevisiae ORC1. Thus, the protein encoded by orp1+ may represent a component of an Sch. pombe ORC. The orp1+ gene is also closely related to an uncharacterized putative human homolog. It is likely that the members of the cdc18/CDC6 family play key roles in the regulation of DNA replication during the cell cycle of diverse species from archaebacteria to man (Muzi-Falconi, 1995).

In a screen for new cell-cycle genes in Schizosaccharomyces pombe has led to the isolation of cdc30, which is identical to orp1, a putative homolog of the Saccharomyces cerevisiae ORC1 gene. Analysis of the temperature-sensitive orp1-4 and the orp1(delta) mutants indicates that orp1 is required at the onset of S phase for an early step of DNA replication. Orp1p is found in the nucleus and is present at a constant level throughout the cell cycle. Genetic interactions occur between orp1 and cdc18 and cdc21 (an MCM homolog). Orp1p forms protein complexes with both cdc18p and cdc21p in vivo, suggesting that interactions between these proteins and ORC are important for controlling the initiation of DNA replication at the onset of S phase. The orp1 gene is also required for the control that prevents entry into mitosis in the absence of DNA replication, suggesting a role for ORC in this checkpoint pathway (Grallert, 1996).

The origin recognition complex (ORC) binds to the well defined origins of DNA replication in budding yeast. Homologous proteins in other eukaryotes have been identified but are less well characterized. The characterization of a fission yeast ORC complex (SpORC) is reported. Database searches identified a fission yeast Orc5 homolog. SpOrc5 is essential for cell viability and its deletion phenotype is identical to that of two previously identified ORC subunit homologs, SpOrc1 (Orp1/Cdc30) and SpOrc2 (Orp2). Co-immunoprecipitation experiments demonstrate that SpOrc1 forms a complex with SpOrc2 and SpOrc5 and gel filtration chromatography shows that SpOrc1 and SpOrc5 fractionate as high molecular mass complexes. SpORC subunits localize to the nucleus in a punctate distribution which persists throughout interphase and mitosis. A chromatin isolation protocol was developed; SpOrc1, 2 and 5 were shown to be associated with chromatin at all phases of the cell cycle. While the levels, nuclear localization and chromatin association of SpORC remain constant through the cell cycle, one of its subunits, SpOrc2, is differentially modified. SpOrc2 is a phosphoprotein which is hypermodified in mitosis and is rapidly converted to a faster migrating isoform as cells proceed into G(1) in preparation for S-phase (Lygerou, 1999).

Saccharomyces cerevisiae Cdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotide-binding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, the conserved Lys114 at the nucleotide-binding site was changed to five other amino acid residues. These mutants were used to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6 temperature-sensitive mutant cells. The results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, its gene functions in DNA replication were analyzed by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants show the complete retraction of DNA synthesis. In order to test its effect on the G1/S transition of the cell cycle, temporal and spatial studies of yeast replication complex were carried out. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. cdc6(K114E) was found to be defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotide-binding function, Cdc6 protein has been demonstrated to associate with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. These results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP-binding of Cdc6; this may be a prerequisite step to assemble the operational replicative complex at the G1/S transition (Wang, 1999).

Silencing of the cryptic mating-type loci HMR and HML requires the recognition of DNA sequence elements called silencers by the Sir1p, one of four proteins dedicated to the assembly of silenced chromatin in Saccharomyces cerevisiae. The Sir1p is thought to recognize silencers indirectly through interactions with proteins that bind the silencer DNA directly, such as the origin recognition complex (ORC). Eight recessive alleles of SIR1 were discovered that encode mutant Sir1 proteins specifically defective in their ability to recognize the HMR-E silencer. The eight missense mutations all map within a 17-amino-acid segment of Sir1p, and this segment was also required for Sir1p's interaction with Orc1p. The mutant Sir1 proteins could function in silencing if tethered to a silencer directly through a heterologous DNA-binding domain. Thus the amino acids identified are required for Sir1 protein's recognition of the HMR-E silencer and interaction with Orc1p, but not for its ability to function in silencing per se. The approach used to find these mutations may be applicable to defining interaction surfaces on proteins involved in other processes that require the assembly of macromolecular complexes (Gardner, 1999).

Replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Evidence is presented that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1(+)/cdc19(+) and mis5(+) genes, respectively, are associated with chromatin DNA only during the G(1) and S phases. Immunostaining of spread nuclei shows SpMcm6p is localized at discrete foci on chromatin during the G(1) and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p is preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p is associated with these origins only in the G(1) and S phases. Both Orp1p and SpMcm6p are associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication (Ogawa, 1999).

An in situ technique is described for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe. After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. This method has been applied to the DNA replication factor mcm4/cdc21; chromatin association is found to occur during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication. Upon overexpressing cdc18, mcm4 is shown to be required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication (Kearsey, 2000).

The origin recognition complex (ORC) binds to the specific positions on chromosomes that serve as DNA replication origins. Although ORC is conserved from yeast to humans, the DNA sequence elements that specify ORC binding are not. In particular, metazoan ORC shows no obvious DNA sequence specificity, whereas yeast ORC binds to a specific DNA sequence within all yeast origins. Yeast origins, named ARSs (autonomous replicating sequences) for their ability to endow autonomous replication to plasmids, contain an ORC-binding site, a bipartite DNA sequence containing a conserved 15-base-pair (bp) A-element, and a smaller, less conserved, B1-element. When yeast ORC binds to its site, it protects ~35 bp of DNA that includes these defined elements. Whereas chromatin must play an important role in metazoan ORC's ability to recognize origins, it is unclear whether chromatin plays a role in yeast ORC's recognition of origins. This study focused on the role of the conserved N-terminal bromo-adjacent homology domain of yeast Orc1 (Orc1BAH). Recent studies indicate that BAH domains are chromatin-binding modules. This study shows that the Orc1BAH domain is necessary for ORC's stable association with yeast chromosomes, and is physiologically relevant to DNA replication in vivo. This replication role is separable from the Orc1BAH domain's previously defined role in transcriptional silencing. Genome-wide analyses of ORC binding in ORC1 and orc1bahDelta cells revealed that the Orc1BAH domain contributes to ORC's association with most yeast origins, including a class of origins highly dependent on the Orc1BAH domain for ORC association (orc1bahDelta-sensitive origins). Orc1bahDelta-sensitive origins require the Orc1BAH domain for normal activity on chromosomes and plasmids, and are associated with a distinct local nucleosome structure. Thus, yeast ORC selects origins through a combination of ORC-DNA and ORC-chromatin interactions, and the extent to which one type of interaction contributes to origin selection varies with individual origins. These data provide molecular insights into how the Orc1BAH domain contributes to ORC's selection of replication origins, as well as new tools for examining conserved mechanisms governing ORC's selection of origins within eukaryotic chromosomes (Müller, 2010).

Regulation of ORC1 expression

The initiation of DNA replication in Saccharomyces cerevisiae requires the action of a multisubunit complex of six proteins known as the origin recognition complex (ORC). The identification of higher eukaryotic homologs of several ORC components suggests a universal role for this complex in DNA replication. The expression of one of these homologs is regulated by cell proliferation. Expression of the human Orc1 gene (HsOrc1) is low in quiescent cells, and it is then dramatically induced upon stimulation of cell growth. In contrast, expression of the HsOrc2 gene does not appear to be similarly regulated. The promoter that regulates HsOrc1 transcription was isolated; the promoter confers cell growth-dependent expression. The cell growth control is largely the consequence of E2F-dependent negative transcription control in quiescent cells. Activation of HsOrc1 transcription following growth stimulation requires G1 cyclin-dependent kinase activity, and forced E2F1 expression can bypass this requirement. These results thus provide a direct link between the initiation of DNA replication and the cell growth regulatory pathway involving G1 cyclin-dependent kinases, the Rb tumor suppressor, and E2F (Ohtani, 1996).

Subunit interactions of ORC and recognition of DNA by ORC

The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologs of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). HsORC has been extracted from HeLa cell chromatin and its subunit composition has been probed using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologs of Orc1-Orc5 but lacks a putative homolog of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, it was possible to identify a complex containing all six subunits. To explore the subunit-subunit interactions that are required for the assembly of HsORC, extensive co-immunoprecipitation experiments were carried out with recombinant ORC subunits expressed in different combinations. These studies reveal the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication (Vashee, 2001).

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe; Sp) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. The N-terminal half of SpOrc4 contains nine AT-hook motifs that specifically bind AT-rich sequences, while the C-terminal half is 35% identical and 63% similar to the human and Xenopus Orc4 proteins. However, while SpOrc4 has a general affinity for all AT-rich DNA, it has a higher affinity for specific asymmetric A:T-rich sequences found within S. pombe replication origins, and initiates bi-directional DNA replication primarily at a single site within the ARS element. Thus, while each AT-hook motif binds tightly to [AAA(T/A)], site specificity probably results from the arrangement of all nine motifs acting in concert. SpOrc4 recruits the remaining five ORC subunits to the DNA-binding site and initiates assembly of a pre-RC. Despite the interspecies ORC differences, ORCs are functionally conserved. (1) SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bind ATP and exhibit ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. (2) SpOrc4, which is solely responsible for binding SpORC to DNA, inhibits up to 70% of Xenopus ORC-dependent DNA replication in Xenopus egg extract by preventing Xenopus ORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 is located at AT-rich sequences. Xenopus ORC in egg extract binds preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruits XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S. pombe (Kong, 2003).

ORC1 only temporarily associates with the ORC complex, only during G1

To investigate the events leading to initiation of DNA replication in mammalian chromosomes, the time when hamster origin recognition complexes (ORCs) became functional was related to the time when Orc1, Orc2 and Mcm3 proteins became stably bound to hamster chromatin. Functional ORCs, defined as those able to initiate DNA replication, are absent during mitosis and early G(1) phase, and reappear as cells progress through G(1) phase. Immunoblotting analysis reveal that hamster Orc1 and Orc2 proteins are present in nuclei at equivalent concentrations throughout the cell cycle, but only Orc2 is stably bound to chromatin. Orc1 and Mcm3 are easily eluted from chromatin during mitosis and early G(1) phase, but become stably bound during mid-G(1) phase, concomitant with the appearance of a functional pre-replication complex at a hamster replication origin. Since hamster Orc proteins are closely related to their human and mouse homologs, the unexpected behavior of hamster Orc1 provides a novel mechanism in mammals for delaying assembly of pre-replication complexes until mitosis is complete and a nuclear structure has formed (Natale, 2000).

The origin recognition complex (ORC) in yeast is a complex of six tightly associated subunits essential for the initiation of DNA replication. Human ORC subunits are nuclear in proliferating cells and in proliferative tissues like the testis, consistent with a role of human ORC in DNA replication. Orc2, Orc3, and Orc5 also are detected in non-proliferating cells like cardiac myocytes, adrenal cortical cells, and neurons, suggesting an additional role of these proteins in non-proliferating cells. Although Orc2-5 co-immunoprecipitate with each other under mild extraction conditions, a holo complex of the subunits is difficult to detect. When extracted under more stringent extraction conditions, several of the subunits co-immunoprecipitate with stoichiometric amounts of other unidentified proteins but not with any of the known ORC subunits. The variation in abundance of individual ORC subunits (relative to each other) in several tissues, expression of some subunits in non-proliferating tissues, and the absence of a stoichiometric complex of all the subunits in cell extracts indicate that subunits of human ORC in somatic cells might have activities independent of their role as a six subunit complex involved in replication initiation. Finally, all ORC subunits remain consistently nuclear, and Orc2 is consistently phosphorylated through all stages of the cell cycle, whereas Orc1 is selectively phosphorylated in mitosis (Thome, 2000).

It is proposed that the full ORC complex, including Orc1, is formed in proliferating cells but only during a small window of time in the cell cycle or in a sub-nuclear fraction that is difficult to extract in lysates. The co-precipitation and gel filtration experiments in 0.1% Nonidet P-40 buffer indicate that at least a sub-complex of Orc2, Orc3, Orc4, and Orc5 can be detected in cell extracts. In addition several of the ORC subunits are detected in non-proliferating cells. Upon induction of the cell cycle, Orc1 is expressed from an E2F-regulated promoter. The newly synthesized Orc1 is better extracted than mature Orc1 but is not associated with other ORC subunits. It is speculate that the new Orc1 protein subsequently associates with the other ORC subunits (most likely in a cellular fraction that is tightly associated with the chromatin) and renders ORC competent for initiating DNA replication. This suggestion is consistent with a recent report that in Chinese hamster ovary cells Orc2 remains firmly associated with chromatin in all phases of the cell cycle, whereas Orc1 selectively associates with chromatin in G1, thereby making the origins competent to replicate (Thome, 2000).

The association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin was investigated in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei show that a complex containing hOrc1p and hOrc2p occurs in a nuclease-resistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. It is proposed that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicate that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle (Kreitz, 2001).

Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Using an in vivo chemical cross-linking method, is has been shown that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells. CDC6 proteins also assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC1 has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. Some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder bind to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. However, the results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed (Fujita, 2002).

The levels of hemagglutinin tagged ORC1 (HA-ORC1) proteins were examined during the cell cycle. A clear decrease was observed in the S-phase cells. This is strikingly different from the previously reported finding that ORC1 protein levels are relatively constant during the cell cycle. The reason for the difference is not clear at present. Because an HA-tagged system was used, the specificity of results with immunoassays could be higher. When MG132, a peptide aldehyde that binds and inactivates 26 S proteasomes, was added to the culture, the S-phase degradation of ORC1 was decreased, and a ladder of ORC1 bands with much retarded mobility, presumably due to ubiquitinylated intermediates, was detected. No obvious difference in MCM7 or ORC2 protein levels was observed among the samples. Together, the available data suggest that human ORC1 proteins are degraded during S phase via the ubiquitin-proteasome system (Fujita, 2002).

The binding regions were examined of components of the origin recognition complex (ORC) in the human genome. For this purpose, chromatin immunoprecipitation assays were performed with antibodies against human Orc1 and Orc2 proteins. A binding region for human Orc proteins 1 and 2 was identified in a <1-kbp segment between two divergently transcribed human genes. The region is characterized by CpG tracts and a central sequence rich in AT base pairs. Both, Orc1 and Orc2 proteins are found at the intergenic region in the G1 phase, but S-phase chromatin contains only Orc2 protein, supporting the notion that Orc1p dissociates from its binding site in the S phase. Sequences corresponding to the intergenic region are highly abundant in a fraction of nascent DNA strands, strongly suggesting that this region not only harbors the binding sites for Orc1 protein and Orc2 protein but also serves as an origin of bidirectional DNA replication (Ladenberger, 2002).

The pre-replication complex in human cells is regulated by the temporal accumulation of ORC1 in G1 nuclei

Components of the origin recognition complex are highly conserved among eukaryotes and are thought to play an essential role in the initiation of DNA replication. The level of the largest subunit of human ORC (ORC1) during the cell cycle was studied in several human cell lines with a specific antibody. In all cell lines, ORC1 levels oscillate: ORC1 starts to accumulate in mid-G1 phase, reaches a peak at the G1/S boundary, and decreases to a basal level in S phase. In contrast, the levels of other ORC subunits (ORCs 2-5) remain constant throughout the cell cycle. The oscillation of ORC1, or the ORC1 cycle, also occurs in cells expressing ORC1 ectopically from a constitutive promoter. Furthermore, the 26 S proteasome inhibitor MG132 blocks the decrease in ORC1, suggesting that the ORC1 cycle is mainly due to 26 S proteasome-dependent degradation. Arrest of the cell cycle in early S phase by hydroxyurea, aphidicolin, or thymidine treatment is associated with basal levels of ORC1, indicating that ORC1 proteolysis starts in early S phase and is independent of S phase progression. These observations indicate that the ORC1 cycle in human cells is highly linked with cell cycle progression, allowing the initiation of replication to be coordinated with the cell cycle and preventing origins from refiring (Tatsumi, 2003).

The origin recognition complex (ORC) plays a central role in regulating the initiation of DNA replication in eukaryotes. The level of the ORC1 subunit oscillates throughout the cell cycle, defining an ORC1 cycle. ORC1 accumulates in G1 and is degraded in S phase, although other ORC subunits (ORCs 2-5) remain at almost constant levels. The behavior of ORC components in human cell nuclei with respect to the ORC1 cycle demonstrates that ORCs 2-5 form a complex that is present throughout the cell cycle and that associates with ORC1 when it accumulates in G1 nuclei. ORCs 2-5 are found in both nuclease-insoluble and -soluble fractions. The appearance of nuclease-insoluble ORCs 2-5 parallels the increase in the level of ORC1 associating with nuclease-insoluble, non-chromatin nuclear structures. Thus, ORCs 2-5 are temporally recruited to nuclease-insoluble structures by formation of the ORC1-5 complex. An artificial reduction in the level of ORC1 in human cells by RNA interference results in a shift of ORC2 to the nuclease-soluble fraction, and the association of MCM proteins with chromatin fractions is also blocked by this treatment. These results indicate that ORC1 regulates the status of the ORC complex in human nuclei by tethering ORCs 2-5 to nuclear structures. This dynamic shift is further required for the loading of MCM proteins onto chromatin. Thus, the pre-replication complex in human cells may be regulated by the temporal accumulation of ORC1 in G1 nuclei (Ohta, 2003).

Mammalian Orc1 protein is selectively released from chromatin and ubiquitinated during the S-to-M transition in the cell division cycle

Changes in the affinity of the Orc1 protein for chromatin in hamsters during the M-to-G(1) transition have been shown to correlate with the activity of hamster origin recognition complexes (ORCs) and the appearance of prereplication complexes at specific sites. Orc1 is shown to be selectively released from chromatin as cells enter S phase, converted into a mono- or diubiquitinated form, and then deubiquitinated and re-bound to chromatin during the M-to-G(1) transition. Orc1 is degraded by the 26S proteasome only when released into the cytosol, and peptide additions to Orc1 make it hypersensitive to polyubiquitination. In contrast, Orc2 remains tightly bound to chromatin throughout the cell cycle and is not a substrate for ubiquitination. Since the concentration of Orc1 remains constant throughout the cell cycle, and its half-life in vivo is the same as that of Orc2, ubiquitination of non-chromatin-bound Orc1 presumably facilitates the inactivation of ORCs by sequestering Orc1 during S phase. Thus, in contrast to yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe), mammalian ORC activity appears to be regulated during each cell cycle through selective dissociation and reassociation of Orc1 from chromatin-bound ORCs (Li, 2002).

The results support the existence of a novel regulatory pathway in the initiation of mammalian DNA replication. In mammals, Orc1 and Orc2 are both tightly bound to chromatin during late G1 phase, and late G1-phase nuclei contain active ORC-chromatin sites by virtue of the fact that they can initiate DNA replication at specific genomic sites when incubated in an Orc-depleted Xenopus egg extract. In contrast to yeast in which all six ORC subunits are stably bound to chromatin throughout the cell cycle, the affinity of mammalian Orc1 for chromatin is selectively reduced during S phase, such that lysis of cells in 0.1% Triton X-100, 0.15 M NaCl, and 1 mM Mg2+-ATP releases Orc1, but not Orc2, into the non-chromatin-bound fraction. Moreover, the Orc1 that is released during S phase is rapidly ubiquitinated (Li, 2002).

Although Orc2 was generally not detected in the non-chromatin-bound fraction, a small amount was sometimes observed. Whether this resulted from chromatin fragmentation or the release of a special Orc1/Orc2-containing complex is not known. However, the amount of Orc2 tightly bound to chromatin remains essentially constant throughout the cell cycle, and neither endogenous Orc2 nor FLAG-Orc2 was a substrate for ubiquitination, suggesting that only Orc1 is released from chromatin-bound ORCs. Orc4p also remains tightly bound to chromatin throughout the cell cycle (Li, 2002).

Since only half of the Orc1 was released during S phase, it is postulated that Orc1 is released only from those ORC-chromatin sites where replication has initiated (old origins). However, since all of the Orc1 in metaphase cells (particularly those cells that have not been arrested in nocodazole) appears in the non-chromatin-bound fraction, the remaining Orc1 must be released from ORCs bound to newly replicated origins when S phase is completed. Since metaphase chromatin cannot initiate DNA replication in Xenopus egg extracts depleted of Xenopus Orc proteins, the missing hamster Orc1 subunit does not allow the remaining hamster Orc proteins to form an active ORC-chromatin site. Therefore, release of hamster Orc1 during S phase would prevent reinitiation of DNA replication at those replication origins (Li, 2002).

What triggers the release of Orc1 from ORC-chromatin sites? Since both Orc1 and Ub-Orc1 are rapidly released from chromatin when cells enter S phase and Ub-Orc1 is largely absent from mitotic cells, ubiquitination per se is not likely the cause of Orc1 release, but it is a mechanism to prevent its reassociation with ORC-chromatin sites during S phase. The affinity of Orc proteins for chromatin may depend on their phosphorylated state. For example, both XlOrc1 and XlOrc2 are hyperphosphorylated in metaphase-arrested eggs relative to activated eggs, and Xenopus ORCs can be selectively released from chromatin by incubating chromatin either in a metaphase extract or with Cdc2/cyclin A. Thus, cyclin-dependent protein kinases may be responsible for altering the affinity of Orc1 for ORC-chromatin sites as cells transit from S to M phase (Li, 2002).

Stable binding of Orc1 to chromatin occurs even when cells are arrested in nocodazole. Therefore, the affinity of Orc1 for chromatin is not linked to microtubule assembly. However, the amount of Orc1 bound to metaphase chromatin is much less than with interphase chromatin, and active ORC-chromatin sites are not detectable in metaphase chromatin, despite the presence of mammalian Cdc6p. Mcm proteins may also begin binding to chromatin during this time period, but they do not produce active pre-RCs because active ORCs and pre-RCs do not appear until after cells have completed mitosis and formed an intact nuclear membrane. In fact, chromatin-bound Cdc6p disappears during early G1 and reappears during late G1, consistent with assembly of functional pre-RCs during late G1 phase (Li, 2002).

Orc1 protein associates with chromatin in mammalian metaphase cells under several conditions (cells permeabilized with digitonin, cells overexpressing Orc1, and cells arrested in TN16 for 24 h). Differences in the amount of Orc1 eluted from chromatin in these experiments may reflect the presence or absence of ATP in the extraction buffer, the length of time cells are arrested in nocodazole, or the use of uncharacterized drugs, such as TN16. Nevertheless, chromatin-bound Orc1 in metaphase cells, relative to other Orc proteins, is salt sensitive, and ORC activity is absent. Therefore, whatever the nature of the association between Orc1 and metaphase chromatin, it does not produce functional ORCs. Restoration of ORC activity reappears only when Orc1 binds tightly to chromatin in early G1-phase cells (Li, 2002).

ORC activity is restored during the 2-h transition from M to G1 phase, concomitant with the appearance of tightly bound Orc1. Moreover, Mcm proteins, which are not bound to metaphase chromatin, begin to reassociate with chromatin during late telophase and reach their maximum level of association by 2 h after release from metaphase, consistent with the assembly of pre-RCs. Thus, the assembly of pre-RCs during the M-to-G1 transition is limited by the rate of assembly of functional ORCs on chromatin; this rate appears to be limited by the rate at which Orc1 becomes tightly bound to chromatin. These events are inversely correlated with the loss of cyclin B, which is required to drive cells into mitosis, and the formation of a nuclear envelope, which is required for the initiation of DNA replication. Loss of cyclin B during the M-to-G1 transition would be expected to alter the phosphorylation pattern of proteins, one of which may be Orc1 (Li, 2002).

A significant portion of the Orc1 that is released from chromatin is modified by ubiquitination. However, the role of ubiquitination during the S-to-M transition is not to destroy Orc1 but to sequester it in order to prevent its reassociation with ORC-chromatin sites. The rate of Orc1 degradation in vivo is the same as that of nonubiquitinated Orc2, and the intracellular concentration of Orc1 remained essentially constant throughout the cell cycle. Since little if any of the Orc1 in mitotic cells is ubiquitinated, Ub-Orc1 must be converted back into unbound Orc1 sometime during the S-to-M transition, suggesting an equilibrium between ubiquitination and deubiqitination of unbound Orc1. Nonubiquitinated Orc1 then rebinds to chromatin during the subsequent G1 phase to produce active ORCs at specific chromosomal sites (Li, 2002).

This conclusion is not surprising in view of the fact that ubiquitination is limited to one or possibly two adducts, whereas efficient degradation of ubiquitinated proteins requires chains of four or more Ub molecules. Despite the fact that Ub-Orc1 migrated during SDS-PAGE as a molecule 20 kDa larger than Orc1 (suggesting two Ub adducts), three other experiments detected only one Ub adduct. (1) Ubiquitination of endogenous Orc1 by GST-Ub produces a single major species whose molecular mass is consistent with only one GST-Ub adduct. (2) Ub hydrolase (an enzyme that specifically removes Ub and not Ub-related proteins such as SUMO-1; see Drosophila SUMO) converts Ub-Orc1 into Orc1, whereas proteasome inhibitors such as CLBL and MG132 allow conversion of Orc1 into the 119-kDa form of Orc1. In both cases, no intermediate-sized proteins are detected, suggesting that the 119-kDa Orc1 contained a single Ub adduct (Li, 2002).

One can envisage at least three advantages to regulating ORC activity in metazoan cells. The first is to prevent reassembly of pre-RCs during S phase by simply inactivating ORC-chromatin sites where initiation occurs through destabilization and subsequent ubiquitination of Orc1. Since mammalian Cdc6p binds Orc1, selective inactivation of Orc1 may directly prevent recruitment of Cdc6p to the ORC-chromatin site. By applying this strategy to newly assembled ORC-chromatin sites as well as to those sites where initiation has occurred, the decision to reenter a proliferative cell cycle is delayed until mitosis is complete and a nuclear membrane has been reassembled. This leads directly to a second regulatory role for Orc1 (Li, 2002).

Reassembly of Orc1 into stable ORC-chromatin sites provides a mechanism for selecting which of the many potential initiation sites along the genome will be activated. Concurrent with the accumulation of stably bound Orc1 and Mcm proteins on mammalian chromatin is the appearance of pre-RCs in nuclei from G1-phase cells at the same specific initiation sites normally used in vivo. This cell cycle-dependent event is referred to as the origin decision point. Prior to the origin decision point, Xenopus egg extract can initiate DNA replication randomly throughout the hamster genome, a phenomenon that is dependent on Xenopus Orc proteins. This phenomenon could account for the observation that both early and late replicating regions of the hamster genome replicate concurrently when early G1 nuclei (1 h postmetaphase) are incubated in a Xenopus egg extract. Thus, the absence of stably bound Orc1 during the S-to-M transition allows the number and locations of initiation sites to be reprogrammed during animal development (Li, 2002). Finally, cell cycle-dependent ORC instability offers a primary checkpoint control mechanism by which cells could disassemble ORC-chromatin sites and thereby prevent proliferation under special circumstances, for example, cells that have sustained DNA damage or that have entered a quiescent or terminally differentiated state. Cells entering a quiescent state have, in fact, been shown to lose their ability to establish pre-RCs at specific genomic sites and to bind Cdc6 and Mcm proteins to their chromatin. Moreover, terminally differentiated cells lack critical proteins required for the assembly of pre-RCs. While the mechanisms involved have not yet been elucidated, ubiquitination and destruction of Orc1 may well trigger a cascade of events that shuts down cell proliferation pathways (Li, 2002).

hOrc1p destruction occurs through the proteasome and is signaled by the SCF(Skp2) ubiquitin-ligase complex

Eukaryotic cells possess overlapping mechanisms to ensure that DNA replication is restricted to the S phase of the cell cycle. The levels of hOrc1p, the largest subunit of the human origin recognition complex, vary during the cell division cycle. In rapidly proliferating cells, hOrc1p is expressed and targeted to chromatin as cells exit mitosis and prereplicative complexes are formed. Later, as cyclin A accumulates and cells enter S phase, hOrc1p is ubiquitinated on chromatin and then degraded. hOrc1p destruction occurs through the proteasome and is signaled in part by the SCF(Skp2) ubiquitin-ligase complex. Other hORC subunits are stable throughout the cell cycle. The regulation of hOrc1p may be an important mechanism in maintaining the ploidy in human cells (Mendez, 2002).

Sequence- specific DNA-binding proteins are involved in recruiting ORC1 to regulate replication initiation and/or transcription repression

The promoter of the rat aldolase B (AldB) gene functions in vivo as an origin of DNA replication in the cells in which transcription of the gene is repressed. Two closely related DNA-binding proteins, AlF-C1 and AlF-C2, repress the AldB gene promoter. The binding site of these proteins, site C, is one of the required DNA elements of the AldB gene origin/promoter for autonomously replicating activity in transfected cells. AlF-C1 and AlF-C2 bind directly to Orc1, a subunit of the origin recognition complex (ORC). Deletion analyses revealed a functional domain in AlF-C2 for binding to Orc1, which is located separately from the DNA-binding domain. In addition, a novel protein-interacting domain in Orc1 required for the binding of AlF-C2, is conserved in human, mouse and Chinese hamster, but not in Drosophila, frog and yeast. Thus, it is assumed that in mammalian cells, sequence- specific DNA-binding proteins are involved in recruiting ORC to regulate replication initiation and/or transcription repression (Saitoh, 2002).

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA

The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. This study established that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, it was shown that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, it was demonstrated that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. This study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation (Samel, 2014).

Other ORC1 interactions

In a two-hybrid screen for proteins that interact with human PCNA, a human protein (hCdc18) homologous to yeast CDC6/Cdc18 and human Orc1 were identified. Unlike yeast, in which the rapid and total destruction of CDC6/Cdc18 protein in S phase is a central feature of DNA replication, the total level of the human protein is unchanged throughout the cell cycle. Epitope-tagged protein is nuclear in G1 and cytoplasmic in S-phase cells, suggesting that DNA replication may be regulated by either the translocation of this protein between the nucleus and the cytoplasm or the selective degradation of the protein in the nucleus. Mutation of the only nuclear localization signal of this protein does not alter its nuclear localization, implying that the protein is translocated to the nucleus through its association with other nuclear proteins. Rapid elimination of the nuclear pool of this protein after the onset of DNA replication and its association with human Orc1 protein and cyclin-cdks supports its identification as human CDC6/Cdc18 protein (Saha, 1998).

The origin recognition complex (ORC) is an initiator protein for DNA replication, but also effects transcriptional silencing in Saccharomyces cerevisiae and heterochromatin function in Drosophila. It is not known, however, whether any of these functions of ORC is conserved in mammals. A novel protein, HBO1 (histone acetyltransferase binding to ORC), has been identified that interacts with human ORC1 protein, the largest subunit of ORC. HBO1 exists as part of a multisubunit complex that possesses histone H3 and H4 acetyltransferase activities. A fraction of the relatively abundant HBO1 protein associates with ORC1 in human cell extracts. HBO1 is a member of the MYST domain family that includes S. cerevisiae Sas2p, a protein involved in control of transcriptional silencing that also has been genetically linked to ORC function. Thus the interaction between ORC and a MYST domain acetyltransferase is widely conserved. Roles are suggested for ORC-mediated acetylation of chromatin in control of both DNA replication and gene expression (Iizuka, 1999).

The minichromosome maintenance (MCM) proteins, together with the origin recognition complex (ORC) proteins and Cdc6, play an essential role in eukaryotic DNA replication through the formation of a pre-replication complex at origins of replication. A yeast two-hybrid screen was used to identify human MCM2-interacting proteins. One of the proteins identified is identical to the ORC1-interacting protein termed HBO1. HBO1 belongs to the MYST family, characterized by a highly conserved C2HC zinc finger and a putative histone acetyltransferase domain. Biochemical studies confirm the interaction between MCM2 and HBO1 in vitro and in vivo. An N-terminal domain of MCM2 is necessary for binding to HBO1, and a C2HC zinc finger of HBO1 is essential for binding to MCM2. A reverse yeast two-hybrid selection was performed to isolate an allele of MCM2 that is defective for interaction with HBO1; this allele was then used to isolate a suppressor mutant of HBO1 that restores the interaction with the mutant MCM2. This suppressor mutation was located in the HBO1 zinc finger. Taken together, these findings strongly suggest that the interaction between MCM2 and HBO1 is direct and mediated by the C2HC zinc finger of HBO1. The biochemical and genetic interactions of MYST family protein HBO1 with two components of the replication apparatus, MCM2 and ORC1, suggest that HBO1-associated HAT activity may play a direct role in the process of DNA replication (Burke, 2001).

Changing localization of proteins bound to a replication origin of human DNA as a function of transit through the cell cycle

The proteins bound in vivo at the human lamin B2 DNA replication origin and their precise sites of binding were investigated along the cell cycle utilizing two novel procedures based on immunoprecipitation following UV irradiation with a pulsed laser light source. In G(1), the pre-replicative complex contains CDC6, MCM3, ORC1 and ORC2 proteins; of these, the post-replicative complex in S phase contains only ORC2; in M phase none of them are bound. The precise nucleotide of binding was identified for the two ORC and the CDC6 proteins near the start sites for leading-strand synthesis; the transition from the pre- to the post-replicative complex is accompanied by a 17 bp displacement of the ORC2 protein towards the start site (Abdurashidova, 2003).

ORC1 and mitotic checkpoints

S-phase and DNA damage checkpoint controls block the onset of mitosis when DNA is damaged or DNA replication is incomplete. It has been proposed that damaged or incompletely replicated DNA generates structures that are sensed by the checkpoint control pathway, although little is known about the structures and mechanisms involved. DNA replication initiation proteins Orp1p and Cdc18p are shown to be required to induce and maintain the S-phase checkpoint in Schizosaccharomyces pombe. The presence of DNA replication structures correlates with activation of the Cds1p checkpoint protein kinase and the S-phase checkpoint pathway. By contrast, induction of the DNA damage pathway is not dependent on Orp1p or Cdc18p. It is proposed that the presence of unresolved replication forks, together with Orp1p and Cdc18p, are necessary to activate the Cds1p-dependent S-phase checkpoint (Murakami, 2002).

Origins of replication are expected to recruit initiation proteins like ORC and Cdc6 in eukaryotes and provide a platform for unwinding DNA. This study tests whether localization of initiation proteins onto DNA is sufficient for origin function. Different components of the ORC complex and Cdc6 stimulate prereplicative complex (pre-RC) formation and replication initiation when fused to the GAL4 DNA-binding domain and recruit to plasmid DNA containing a tandem array of GAL4-binding sites. Replication occurs once per cell cycle and is inhibited by Geminin, indicating that the plasmid is properly licensed during the cell cycle. The GAL4 fusion protein recruits other polypeptides of the ORC-Cdc6 complex, and nascent strand abundance is highest near the GAL4-binding sites. Therefore, the artificial origin recapitulates many of the regulatory features of physiological origins and is valuable for studies on replication initiation in mammalian cells. The utility of this system is demonstrated by showing the functional importance of the ATPase domains of human Cdc6 and Orc1 and the dispensability of the N-terminal segments of Orc1 and Orc2 in this assay. Artificial recruitment of a eukaryotic cellular replication initiation factor to a DNA sequence can create a functional origin of replication, providing a robust genetic assay for these factors and a novel approach to generating episomal vectors for gene therapy (Takeda, 2005).

Previous studies have suggested that the activity of the mammalian origin recognition complex (ORC) is regulated by cell-cycle-dependent changes in its Orc1 subunit. This study shows that Orc1 modifications such as mono-ubiquitylation and hyperphosphorylation that occur normally during S and G2-M phases, respectively, can cause Orc1 to accumulate in the cytoplasm. This would suppress reassembly of pre-replication complexes until mitosis is complete. In the absence of these modifications, transient expression of Orc1 rapidly induces p53-independent apoptosis, and Orc1 accumulates perinuclearly rather than uniformly throughout the nucleus. This behavior mimics the increased concentration and perinuclear accumulation of endogenous Orc1 in apoptotic cells that arise spontaneously in proliferating cell cultures. Remarkably, expression of Orc1 in the presence of an equivalent amount of Orc2, the only ORC subunit that does not induce apoptosis, prevents induction of apoptosis and restores uniform nuclear localization of Orc1. This would promote assembly of ORC-chromatin sites, such as occurs during the transition from M to G1 phase. These results provide direct evidence in support of the regulatory role proposed for Orc1, and suggest that aberrant DNA replication during mammalian development could result in apoptosis through the appearance of 'unmodified' Orc1 (Saha, 2006).

The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo

Selection of initiation sites for DNA replication in eukaryotes is determined by the interaction between the origin recognition complex (ORC) and genomic DNA. In mammalian cells, this interaction appears to be regulated by Orc1, the only ORC subunit that contains a bromo-adjacent homology (BAH) domain. Since BAH domains mediate protein-protein interactions, the human Orc1 BAH domain was mutated, and the mutant proteins expressed in human cells to determine their affects on ORC function. The BAH domain was not required for nuclear localization of Orc1, association of Orc1 with other ORC subunits, or selective degradation of Orc1 during S-phase. It does, however, facilitate reassociation of Orc1 with chromosomes during the M to G1-phase transition, and it is required for binding Orc1 to the Epstein-Barr virus oriP and stimulating oriP-dependent plasmid DNA replication. Moreover, the BAH domain affects Orc1's ability to promote binding of Orc2 to chromatin as cells exit mitosis. Thus, the BAH domain in human Orc1 facilitates its ability to activate replication origins in vivo by promoting association of ORC with chromatin (Noguchi, 2006).

Eukaryotic DNA replication initiates at a large number of chromosomal origins, controlled by the ordered assembly of multiprotein replication complexes and the cell cycle-dependent activity of kinases that phosphorylate them. In cases where origins have been transposed to other chromosomal locations, they have been found to colocalize with genetically defined replicators, i.e., sequences capable of promoting DNA replication at ectopic genomic sites. In metazoan systems, replication origins or replicators are bound by homologues of proteins first characterized for the yeast Saccharomyces cerevisiae, suggesting that the basic mechanisms controlling replication initiation are conserved among eukaryotes. In S. cerevisiae, replicators typically comprise a binding site for the hexameric origin recognition complex ORC and a DNA unwinding element (DUE). ORC enables the Cdc6-, Cdt1-dependent recruitment of the MCM helicase complex to replication origins, forming a prereplication complex (pre-RC) early during the G1 phase of the cell cycle. Cyclin-dependent kinase and DDK activities promote the binding of Mcm10, Cdc45, and RPA to form preinitiation complexes and unwind the DNA template in advance of replication. The effect of kinase activity on the pre-RC is partially to disassemble ORC and release MCMs and Cdc6 from chromatin (Noguchi, 2006 and references therein).

The 2.4-kb 5' region of the human c-myc gene contains multiple transcription factor binding sites and a DUE that is unwound in vivo. The DUE is situated in a 100-bp zone containing three 10/11 matches to the S. cerevisiae ARS consensus sequence. It was initially reported that replication initiates in this region, and quantitative PCR (qPCR) has been used to define the replication initiation zone. Subsequent work has confirmed that replication initiates in the 5' flanking DNA of the c-myc gene in multiple species. The 2.4-kb c-myc core origin endows plasmids with ARS activity in vitro and shows replicator activity when moved to an ectopic chromosomal location. This region displays an ordered chromatin structure stable to chromosomal translocation, and mutational analyses have identified regions of the replicator essential for replication initiation, including the DUE (Noguchi, 2006 and references therein).

Chromatin immunoprecipitation (ChIP) was used in this work to show that the human analogs of the yeast ORC, MCM, and Cdc6 proteins bind preferentially and selectively to the c-myc replicator. The distributions of Mcm3 and Mcm7 are similar in asynchronous cells, with the greatest ChIP signal at, and upstream of, the DNA unwinding element. These distributions change in parallel in cells synchronized in G1 or M phases. By contrast, Orc1, Orc2, and Cdc6 appear to be least abundant at the DUE and each displays a different temporal pattern of replicator binding. The DNA unwinding element binding protein DUE-B, identified using the c-myc DUE as bait in a yeast one-hybrid assay, preferentially binds near the c-myc DUE in a pattern comparable to that of the MCMs in asynchronous and G1-phase cells. Furthermore, at an ectopic locus, c-myc replicator deletions that removed the DUE or altered chromatin structure suppressed DUE-B or Mcm3 binding, respectively, and eliminated origin activity. The relationship between chromatin structure, MCM binding, and origin activity is supported by the demonstration that inhibition of histone deacetylase activity by trichostatin A (TSA) causes a redistribution of Mcm3 binding similar to the broadening of the c-myc replication initiation zone. These results suggest that pre-RC proteins bind nonrandomly to the c-myc replicator and that c-myc origin activity is a function of ORC, MCM, Cdc6, and DUE-B binding to c-myc chromatin (Noguchi, 2006).

Origin recognition complex subunit 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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