A Potential Role for Mini-chromosome Maintenance (MCM) Proteins in Initiation at the Dihydrofolate Reductase Replication Origin
2002; Elsevier BV; Volume: 277; Issue: 4 Linguagem: Inglês
10.1074/jbc.m108118200
ISSN1083-351X
AutoresMark G. Alexandrow, Marion Ritzi, Alexander Pemov, Joyce L. Hamlin,
Tópico(s)Chromosomal and Genetic Variations
ResumoMini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-β, ori-β′, and ori-γ) being preferred. We show here that antibodies to human MCMs 2–7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronizedcultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFRdomain, suggesting a dynamic interaction with DNA. Inasynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells. Mini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-β, ori-β′, and ori-γ) being preferred. We show here that antibodies to human MCMs 2–7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronizedcultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFRdomain, suggesting a dynamic interaction with DNA. Inasynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells. dihydrofolate reductase origin recognition complex mini-chromosome maintenance chromatin immunoprecipitation phosphate-buffered saline Chinese hamster ovary Although much is known about the mechanism of initiation at the origins of mammalian viruses, little is known about these processes at mammalian chromosomal origins of replication. In viral, yeast, and bacterial replicons, initiation is confined to genetically definedreplicator sequences, which direct the loading of initiation factors followed by localized melting of adjacent DNA sequences (reviewed in Ref. 1Kornberg A. Baker T.A. DNA Replication.2nd Ed. W. H. Freeman and Company, New York1992Google Scholar). This interaction allows access to the origin by primases, polymerases, and other factors involved in the elongation steps of replication. In these relatively simple systems, the termorigin is often used to refer to both the replicator (initiation protein binding site) and the local site(s) where nascent strand synthesis begins.Considerable evidence suggests that initiation at mammalian origins is much more complex. A case in point is the origin in the Chinese hamsterDHFR1 domain, which lies in the spacer region between the DHFR and2BE2121 genes (Fig. 1) (2Heintz N.H. Hamlin J.L. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4083-4087Crossref PubMed Scopus (120) Google Scholar, 3Leu T.-H. Hamlin J.L. Mol. Cell. Biol. 1989; 9: 523-531Crossref PubMed Scopus (95) Google Scholar, 4Vaughn J.P. Dijkwel P.A. Hamlin J.L. Cell. 1990; 61: 1075-1087Abstract Full Text PDF PubMed Scopus (195) Google Scholar) and which has been analyzed by almost all of the available origin mapping techniques for localizing nascent strand initiation sites (reviewed in Refs. 5Dijkwel P.A. Hamlin J.L. J. Cell. Biochem. 1996; 62: 210-222Crossref PubMed Scopus (16) Google Scholar and 6DePamphilis M.L. Methods. 1997; 13: 211-219Crossref PubMed Scopus (27) Google Scholar). It was expected that start sites would lie close to replicator sequences that bind to proteins required for initiation. However, these mapping studies have shown that replication can initiate at any one of a very large number of potential sites distributed throughout the 55-kb spacer (4Vaughn J.P. Dijkwel P.A. Hamlin J.L. Cell. 1990; 61: 1075-1087Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 7Dijkwel P.A. Hamlin J.L. Mol. Cell. Biol. 1995; 15: 3023-3031Crossref PubMed Scopus (91) Google Scholar, 8Dijkwel P.A. Hamlin J.L. Mol. Cell. Biol. 1992; 12: 3715-3722Crossref PubMed Scopus (111) Google Scholar), with at least three subregions (termed ori-β, ori-β′, and ori-γ) being preferred (3Leu T.-H. Hamlin J.L. Mol. Cell. Biol. 1989; 9: 523-531Crossref PubMed Scopus (95) Google Scholar,9Kobayashi T. Rein T. 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Nature. 1992; 357: 128-134Crossref PubMed Scopus (988) Google Scholar). ORC binds to origins throughout the cell cycle and helps to recruit other initiation factors in a stepwise manner (11Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar, 13Romanowski P. Madine M.A. Rowles A. Blow J.J. Laskey R.A. Curr. Biol. 1996; 6: 1416-1425Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 14Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 15Coleman T.R. Carpenter P.B. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 16Rowles A. Chong J.P.J. Brown L. Howell M. Evan G.I. Blow J.J. Cell. 1996; 87: 287-296Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). During G1 phase, the Cdc6 protein is synthesized and localized to origins via interaction with ORC (11Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar, 14Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 15Coleman T.R. Carpenter P.B. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 17Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). Recruitment of Cdc6, in turn, facilitates recruitment of the mini-chromosome maintenance (MCM) complex (11Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar, 13Romanowski P. Madine M.A. Rowles A. Blow J.J. Laskey R.A. Curr. Biol. 1996; 6: 1416-1425Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 14Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 15Coleman T.R. 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Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 16Rowles A. Chong J.P.J. Brown L. Howell M. Evan G.I. Blow J.J. Cell. 1996; 87: 287-296Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 17Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 20Diffley J.F.X. Cocker J.H. Dowell S.J. Rowley A. Cell. 1994; 78: 303-316Abstract Full Text PDF PubMed Scopus (466) Google Scholar, 21Diffley J.F.X. Cocker J.H. Nature. 1992; 357: 169-172Crossref PubMed Scopus (292) Google Scholar). Loss-of-function mutations in any of the ORC, MCM, or cdc6 genes in yeast impairs replication and plasmid stability, and reduces the level of initiation occurring at individual origins, suggesting important roles for each of these factors in the initiation reaction (reviewed in Ref. 11Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar).Although mammalian origins are clearly more complex than those of yeast, homologues of many of the proteins involved in initiation in yeast have been identified in metazoans. Counterparts of all six ORC subunits have been identified in fruit flies (22Gossen M. Pak D.T.S. Hansen S.K. Acharya J.K. Botchan M.R. Science. 1995; 270: 1674-1677Crossref PubMed Scopus (128) Google Scholar, 23Chesnokov I. Gossen M. Remus D. Botchan M. Genes Dev. 1999; 13: 1289-1296Crossref PubMed Scopus (87) Google Scholar), several subunits have been found in humans (24Gavin K.A. Hidaka M. Stillman B. Science. 1995; 270: 1667-1671Crossref PubMed Scopus (202) Google Scholar, 25Quintana D.G. Hou Z.-H. Thome K.C. Hendricks M. Saha P. Dutta A. J. Biol. Chem. 1997; 272: 28247-28251Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 26Quintana D.G. Thome K.C. Hou Z.-H. Ligon A.H. Morton C.C. Dutta A. J. Biol. 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Altogether, these data suggest that at least some of the mechanisms involved in identifying and preparing mammalian origins for initiation are likely to be conserved among eukaryotes.Therefore, it is likely that mammalian cells rely, at least in part, on the formation and proper regulation of pre-RC complexes at or near chromosomal origins of replication, and that yeast homologues of ORC, Cdc6, and MCM proteins perform one or more roles in this process. However, it has not yet been shown that any of these proteins functions in mammalian cells in a manner consistent with their suggested roles in initiation. While there is still no functional assay for ORC and Cdc6 in yeast or any other system, some progress has been made in understanding MCM function during initiation and elongation. For example, recent biochemical evidence suggests that the MCM complex may possess helicase activity (38You Z. Komamura Y. Ishimi Y. Mol. Cell. Biol. 1999; 19: 8003-8015Crossref PubMed Scopus (171) Google Scholar, 39Chong J.P. Hayashi M.K. Simon M.N. Xu R.M. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1530-1535Crossref PubMed Scopus (259) Google Scholar, 40Labib K. Tercero J.A. Diffley J.F. Science. 2000; 288: 1643-1647Crossref PubMed Scopus (519) Google Scholar, 41Kelman Z. Lee J.K. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14783-14788Crossref PubMed Scopus (196) Google Scholar, 42Lee J.K. Hurwitz J. J. Biol. Chem. 2000; 275: 18871-18878Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). In addition, in vivocross-linking studies in yeast have shown that the MCM complex binds to origins of replication at the beginning of S-phase, but then migrates with the replication fork after initiation (14Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 40Labib K. Tercero J.A. Diffley J.F. Science. 2000; 288: 1643-1647Crossref PubMed Scopus (519) Google Scholar), consistent with a functional role as a helicase.In this study, we have asked whether MCM subunits are localized preferentially to the DHFR origin region in vivoand whether it is possible to detect any alterations in MCM-chromosome interactions and dynamics that may suggest a functional role for MCMs in replication initiation. We have treated CHO cells with formaldehyde to fix initiation proteins on, or adjacent to, regions of the chromosome where the proteins were situated in vivo. Using this chromatin immunoprecipitation (ChIP) approach, we find that mammalian MCM subunits are preferentially associated with theDHFR origin during the G1/S transition, but become distributed throughout the origin and flanking genes as cells proceed out of S-phase and lose synchrony. These data are consistent with the hypothesis that MCMs localize to this origin during initiation at the beginning of S-phase, and then proceed away from origins with the replication forks.DISCUSSIONUsing antibodies to the human proteins, we have been able to show in Western blots that all six MCM subunits are present in Chinese hamster cells. With regard to interactions among the MCMs in hamster cells, we observed a subcomplex consisting of MCM2, -3, and -5. Similar interactions have been demonstrated in humans and Xenopus(reviewed in Ref. 11Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar, 32Prokhorova T.A. Blow J.J. J. Biol. Chem. 2000; 275: 2491-2498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, and 33Schulte D. Richter A. Burkhart R. Musahl C. Knippers R. Eur. J. Biochem. 1996; 235: 144-151Crossref PubMed Scopus (47) Google Scholar). However, we were not able to detect co-precipitation of MCM4, -6, and -7 with antibodies to any of the three subunits even though the three have been demonstrated to form a three-membered complex in Xenopus and humans (11Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar, 33Schulte D. Richter A. Burkhart R. Musahl C. Knippers R. Eur. J. Biochem. 1996; 235: 144-151Crossref PubMed Scopus (47) Google Scholar, 38You Z. Komamura Y. Ishimi Y. Mol. Cell. Biol. 1999; 19: 8003-8015Crossref PubMed Scopus (171) Google Scholar). Additionally, all six MCMs have been shown to exist as a single large complex in Xenopus and humans (32Prokhorova T.A. Blow J.J. J. Biol. Chem. 2000; 275: 2491-2498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 33Schulte D. Richter A. Burkhart R. Musahl C. Knippers R. Eur. J. Biochem. 1996; 235: 144-151Crossref PubMed Scopus (47) Google Scholar, 48Blow J.J. Tada S. Nature. 2000; 404: 560-561Crossref PubMed Scopus (18) Google Scholar). Since we were not able to demonstrate the latter two complexes here, it is likely that our method of extract preparation differs in subtle ways from those employed in other studies, or that our antisera may disrupt the holocomplex or subcomplexes of MCM proteins.In the ChIP experiments themselves, we observed preferential binding of MCM5 and, to a lesser extent, MCM2, to the intergenic DHFRorigin when cells were arrested at the G1/S boundary (Fig.6). As cells progressed through the S-period, the levels of MCMs in the two flanking genes appeared to increase, but the relative amount of binding to the intergenic region did not diminish substantially. By analogy to yeast (14Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar), if the DHFR origin were 100% efficient (i.e. fired in every cell cycle), it would be expected to be cleared of MCMs as they progress away from the origin with the replication forks once initiation in the region ceases (∼2 h after entry into the S-phase; Ref. 8Dijkwel P.A. Hamlin J.L. Mol. Cell. Biol. 1992; 12: 3715-3722Crossref PubMed Scopus (111) Google Scholar). However, this origin fires in only 15–20% of the amplicons in any one S-period, with the consequence that 80–85% of the origins are replicated passively at later times in S-phase by forks from distant active origins (8Dijkwel P.A. Hamlin J.L. Mol. Cell. Biol. 1992; 12: 3715-3722Crossref PubMed Scopus (111) Google Scholar, 60Dijkwel P.A. Vaughn J.P. Hamlin J.L. Nucleic Acids Res. 1994; 22: 4989-4996Crossref PubMed Scopus (45) Google Scholar). Thus, the MCMs should be concentrated over the intergenic region in early S-phase when 15% of them are active, but should become more uniformly distributed when the cells near the end of S-phase, or in an unsynchronized cell population containing cells at all stages of the cycle. These predictions were, by in large, borne out by the ChIP data on synchronized cells presented in Fig. 6. In chromatin isolated from unsynchronized cultures, MCM5 appears to be relatively uniformly distributed throughout the DHFR region, with no apparent preference for the origin region or the genes. That the ChIP assay is giving a reasonably adequate representation of thein vivo situation is bolstered by preferential binding of the splicing factor PSF-1 to the DHFR and 2BE2121genes in unsynchronized cells (Fig. 5C). However, there was also considerable binding of PSF-1 to cosmids S21 and SE24, which are completely contained within the intergenic region. Possibly, this results from the fact that, like many other genes (61Aranda A. Proudfoot N.J. Mol. Cell. Biol. 1999; 19: 1251-1261Crossref PubMed Google Scholar, 62Cuello P. Boyd D.C. Dye M.J. Proudfoot N.J. Murphy S. EMBO J. 1999; 18: 2867-2877Crossref PubMed Scopus (50) Google Scholar, 63Yonaha M. Proudfoot N.J. Mol. Cell. 1999; 3: 593-600Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), elongating transcription complexes and their associated splicing complexes (64Proudfoot N. Trends Biochem. Sci. 2000; 25: 290-293Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) continue for some distance beyond the polyadenylation sites in theDHFR and 2BE2121genes. 4A. Pemov and J. L. Hamlin, unpublished observations.Other studies in higher eukaryotic organisms have suggested that, while localized to the nucleus throughout most of the cell cycle, MCM proteins are tightly associated with chromatin only in G1and early S-phase, and gradually dissociate from chromatin as cells progress into late S-phase and G2 (11Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar, 13Romanowski P. Madine M.A. Rowles A. Blow J.J. Laskey R.A. Curr. Biol. 1996; 6: 1416-1425Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 35Kimura H. Takizawa N. Nozaki N. Sugimoto K. Nucleic Acids Res. 1995; 23: 2097-2104Crossref PubMed Scopus (67) Google Scholar, 65Hendrickson M. Madine M. Dalton S. Gautier J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12223-12228Crossref PubMed Scopus (104) Google Scholar, 66Kubota Y. Mimura S. Nishimoto S.-I. Takisawa H. Nojima H. 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The solubilized proteins fractionate with the cytoplasmic supernatant, while the insoluble, detergent-resistant protein fractions fractionate with the nuclear pellet, otherwise referred to as the “chromatin pellet” (67Todorov I.T. Attaran A. Kearsey S.E. J. Cell Biol. 1995; 129: 1433-1445Crossref PubMed Scopus (204) Google Scholar, 68Fujita M. Kiyono T. Hayashi Y. Ishibashi M. J. Biol. Chem. 1996; 271: 4349-4354Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 69Donovan S. Harwood J. Drury L.S. Diffley J.F.X. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5611-5616Crossref PubMed Scopus (429) Google Scholar). However, the formaldehyde cross-linking approach covalently fixes chromatin-associated proteins in situ, thereby preventing any potential detergent-soluble proteins from being released from theirin vivo binding sites during cell extract preparation. In light of this difference, our data suggest that at least some MCM proteins are associated with chromatin (and the DHFR region) even during late S-phase and G2, but in a somewhat different biochemical state than in G1 and early S-phase (detergent-sensitive versus -resistant, respectively).We had originally anticipated that there would be little or no MCM interactions with genomic DNA over the two genes (DHFR and2BE2121) adjacent to the intergenic origin region. Thus, the genes were expected to serve as negative controls in samples isolated at the G1/S boundary. The significant amounts of MCM2 and MCM5 detected in the genes therefore could represent nonspecific background cross-linking. In fact, the 2-fold differences that we detect between the genes and the intergenic region at the G1/S boundary are similar to results obtained in virtually all published ChIP assays utilizing PCR approaches in yeast (14Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 49Megee P.C. Mistrot C. Guacci V. Koshland D. Mol. Cell. 1999; 4: 445-450Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar,50Kuo M.-H. Baur E. Struhl K. Allis C.D. Mol. Cell. 2000; 6: 1309-1320Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) and mammalian cell systems (51Cheung P. Tanner K.G. Cheung W.L. Sassone-Corsi P. Denu J.M. Allis C.D. Mol. Cell. 2000; 5: 1-20Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). In these studies as well, only 2–3-fold differences were observed in histone, transcription factor, or DNA replication factor binding to specific versusnonspecific genomic DNA sequences.However, there is another interpretation. Recent work from the Bentley laboratory (70Yankulov K. Todorov I. Romanowski P. Licatalosi D. Cilli K. McCracken S. Laskey R. Bentley D.L. Mol. Cell. Biol. 1999; 19: 6154-6163Crossref PubMed Scopus (72) Google Scholar) has shown that the MCM complex copurifies with RNA polymerase II and general transcription factors in high molecular weight complexes. This group went on to show that the interaction is specific and occurs through the carboxyl-terminal domain of RNA polymerase II. In addition, another group has shown that at least one subunit of the MCM complex interacts with the retinoblastoma protein in mammalian
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