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• W1991658906 abstract "The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G1 and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G1. MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G2, reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G2, at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G1 followed by a recruitment to multiple sites in chromatin subsequent to replication. The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G1 and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G1. MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G2, reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G2, at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G1 followed by a recruitment to multiple sites in chromatin subsequent to replication. Members of the minichromosome maintenance (MCM) 1The abbreviations used are: MCM, minichromosome maintenance; pre-RC, pre-replication complex; ORC, origin recognition complex; FACS, fluorescence-activated cell sorting; ChIP, chromosomal immunoprecipitation; re-ChIP, re-precipitation; PBS, phosphate-buffered saline; DAB, diaminobenzidine tetrahydrochloride; nt, nucleotide(s); GFP, green fluorescent protein.1The abbreviations used are: MCM, minichromosome maintenance; pre-RC, pre-replication complex; ORC, origin recognition complex; FACS, fluorescence-activated cell sorting; ChIP, chromosomal immunoprecipitation; re-ChIP, re-precipitation; PBS, phosphate-buffered saline; DAB, diaminobenzidine tetrahydrochloride; nt, nucleotide(s); GFP, green fluorescent protein. protein family were first characterized in Saccharomyces cerevisiae as essential for plasmid maintenance during the cell cycle (reviewed in Refs. 1Kearsey S.E. Labib K. Biochim. Biophys. Acta. 1998; 1398: 113-136Crossref PubMed Scopus (226) Google Scholar, 2Tye B.K. Annu. Rev. Biochem. 1999; 68: 649-686Crossref PubMed Scopus (499) Google Scholar, 3Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1385) Google Scholar). Each of the six proteins MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7 has a counterpart in yeast, and each is highly conserved throughout eukaryotes. Deletion of genes encoding any one of these six are lethal in S. cerevisiae or Schizosaccharomyces pombe (3Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1385) Google Scholar, 4Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (333) Google Scholar), and these proteins have been ascribed as essential for initiation of DNA replication (5Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar, 6Labib K. Kearsey S.E. Diffley J.F. Mol. Biol. Cell. 2001; 12: 3658-3667Crossref PubMed Scopus (117) Google Scholar). These proteins have been co-isolated as a single complex, and complexes of various combinations of these proteins have been implicated in licensing DNA for initiation of replication in Xenopus egg extracts (7Chong J.P. Mahbubani H.M. Khoo C.Y. Blow J.J. Nature. 1995; 375: 418-421Crossref PubMed Scopus (310) Google Scholar, 8Madine M.A. Khoo C.Y. Mills A.D. Laskey R.A. Nature. 1995; 375: 421-424Crossref PubMed Scopus (231) Google Scholar). Another MCM protein, MCM10, is reportedly required for phosphorylation of components of the MCM2–7 complex prior to initiation (9Lee J.K. Seo Y.S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2334-2339Crossref PubMed Scopus (74) Google Scholar) and has also been implicated in the transition from initiation to replication (10Kawasaki Y. Hiraga S. Sugino A. Genes Cells. 2000; 5: 975-989Crossref PubMed Scopus (66) Google Scholar). Preceding initiation of replication, and at a point near the end of mitosis (11Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar), assembly of the MCM proteins into a prereplication complex (pre-RC) occurs dependent on coordinated function of the origin recognition complex (ORC) (12Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (983) Google Scholar) and proteins Cdc6 (5Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar) and Cdt1 (13Nishitani H. Lygerou Z. Nishimoto T. Nurse P. Nature. 2000; 404: 625-628Crossref PubMed Scopus (365) Google Scholar). Initiation then depends upon activation by cyclin-dependent kinases (14Zou L. Stillman B. Science. 1998; 280: 593-596Crossref PubMed Scopus (273) Google Scholar) and the Dbf4-dependent kinase Cdc7 (15Sclafani R.A. J. Cell Sci. 2000; 113: 2111-2117Crossref PubMed Google Scholar). Evidence derived from temperature-sensitive MCM protein degradation during S-phase (16Labib K. Tercero J.A. Diffley J.F. Science. 2000; 288: 1643-1647Crossref PubMed Scopus (515) Google Scholar) and from measurements of MCM protein distribution in chromatin (5Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar) suggests that in S. cerevisiae, upon transition from initiation to elongation, the MCM2–7 complex distributes with replication forks. MCM presence at replication forks remains controversial, however, because no data directly colocalizes an MCM protein with fork proteins. Although there is evidence that MCM proteins play an important role in DNA replication in mammalian cells, evidence regarding an essential role for any individual MCM protein or a functional role for any potential MCM complex remains elusive. Studies of distribution of MCM proteins in chromatin during the cell cycle indicate certain similarities with the yeast model regarding formation of a potential pre-replication complex. MCM proteins 2, 3, 4, and 5 have been reported to enter chromatin late in mitosis, and the MCM proteins alternate between soluble and chromatin-bound forms (17Mendez J. Stillman B. Mol. Cell. Biol. 2000; 20: 8602-8612Crossref PubMed Scopus (744) Google Scholar). A subset of the MCM2–7 complex, consisting of MCMs 4, 6, and 7 (hereafter referred to as “MCM4,6,7”), has been reported to possess DNA helicase activity (18Lee J.K. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 54-59Crossref PubMed Scopus (157) Google Scholar, 19Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). As isolated from HeLa cells, helicase activity of the MCM4,6,7 complex could be inhibited by Cdk-dependent phosphorylation of MCM4 (20Ishimi Y. Komamura-Kohno Y. You Z. Omori A. Kitagawa M. J. Biol. Chem. 2000; 275: 16235-16241Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 21Ishimi Y. Komamura-Kohno Y. J. Biol. Chem. 2001; 276: 34428-34433Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The MCM4,6,7 complex can be visualized by electron microscopy as a double heterotrimeric ring structure, similar to ring structures of other known helicases, including one of an MCM-like protein from archaebacteria (22Chong 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 (258) Google Scholar). The helicase activity of an MCM4,6,7 complex, reconstituted from recombinant S. pombe proteins, reportedly requires the presence of forked DNA structures (18Lee J.K. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 54-59Crossref PubMed Scopus (157) Google Scholar). Although results taken together suggest that the MCM4,6,7 complex could represent a major replicative helicase, there is little direct evidence for this. In particular, it is not known whether this complex selectively assembles at initiation zones of DNA replication in mammalian cells. Recent reports have dealt with cell cycle distribution of MCM proteins in chromatin in the hamster DHFR locus (23Alexandrow M.G. Ritzi M. Pemov A. Hamlin J.L. J. Biol. Chem. 2002; 277: 2702-2708Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 24Schaarschmidt D. Ladenburger E.M. Keller C. Knippers R. Nucleic Acids Res. 2002; 30: 4176-4185Crossref PubMed Scopus (54) Google Scholar), but these did not specifically address MCM4,6,7 localization or requirements for pre-RC assembly proteins. Another recent paper has examined the cell cycle levels of ORC proteins during the HeLa cell cycle, finding that ORC1 undergoes dynamic changes while the other ORC proteins remain at constant levels (25Ohta S. Tatsumi Y. Fujita M. Tsurimoto T. Obuse C. J. Biol. Chem. 2003; 278: 41535-41540Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). This study did not examine MCM protein locations. Recent reports indicate that certain MCM proteins (26Dziak R. Leishman D. Radovic M. Tye B.K. Yankulov K. J. Biol. Chem. 2003; 278: 27372-27381Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 27DaFonseca C.J. Shu F. Zhang J.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3034-3039Crossref PubMed Scopus (68) Google Scholar), including MCM7 (28Fitch M.J. Donato J.J. Tye B.K. J. Biol. Chem. 2003; 278: 25408-25416Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), may play a role in transcription. In this study we assess the localization and distribution of components of the MCM4,6,7 complex, their clamp-loading partner Cdc6, and their partner in the pre-RC complex, ORC1, in a well characterized initiation zone centered upstream of the c-MYC gene during the HeLa cell cycle. This zone was originally mapped in this laboratory (29Vassilev L. Johnson E.M. Mol. Cell. Biol. 1990; 10: 4899-4904Crossref PubMed Scopus (128) Google Scholar) and subsequently refined in a variety of studies (30Berberich S. Trivedi A. Daniel D.C. Johnson E.M. Leffak M. J. Mol. Biol. 1995; 245: 92-109Crossref PubMed Scopus (43) Google Scholar, 31Dhar S.K. Yoshida K. Machida Y. Khaira P. Chaudhuri B. Wohlschlegel J.A. Leffak M. Yates J. Dutta A. Cell. 2001; 106: 287-296Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 32Ishimi Y. Matsumoto K. Ohba R. Mol. Cell. Biol. 1994; 14: 6489-6496Crossref PubMed Scopus (17) Google Scholar, 33Liu G. Malott M. Leffak M. Mol. Cell. Biol. 2003; 23: 1832-1842Crossref PubMed Scopus (69) Google Scholar). We report a highly selective localization of MCM proteins on chromosomal DNA in a particular segment of this zone together with Cdc6 and ORC1 in late G1-phase. During ensuing S- and G2-phases MCM7, an integral component of this complex, remains at the initial site while Cdc6 together with MCM7, but not ORC1, distribute to distant sites, indicating a recruitment of additional MCM proteins to chromatin during and after replication. Cell Culture and Cell Cycle Synchrony—HeLa cells were maintained in spinner culture in Eagle's Minimal Essential Medium for suspension cultures (with l-glutamine and without calcium and magnesium, Cellgro Mediatech) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2, 95% air. Cell cycle synchrony was achieved using a double thymidine block, exactly as described previously (34Karn J. Johnson E.M. Vidali G. Allfrey V.G. J. Biol. Chem. 1974; 249: 667-677Abstract Full Text PDF PubMed Google Scholar). By this method cells are blocked at the beginning of S-phase. Cell cycle progression was verified by FACS analysis (Fig. 1A), and timing of mitosis was verified by microscopic determination of the mitotic index. To specifically examine time points at mitosis the double thymidine block was employed in conjunction with the microtubule disrupter, nocodazole. Eight hours after release from the double thymidine block, the HeLa cells were treated with nocodazole (methyl-[5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl] carbamate, 100 ng/ml, Sigma-Aldrich) for 4 h. This treatment blocks the already synchronized cells at the G2-M boundary (35Luduena R.F. Roach M.C. Pharmacol. Ther. 1991; 49: 133-152Crossref PubMed Scopus (194) Google Scholar). At that time the cells were released from nocodazole into new medium, and time points were taken for analysis at 1-h intervals. Chromatin Immunoprecipitation (ChIP) and Re-precipitation (reChIP) to Detect Proteins Bound to DNA Sequences Upstream of the c-MYC Gene—Approximately 2 × 107 HeLa cells were collected every 2 or 2.5 h after release from double thymidine block (34Karn J. Johnson E.M. Vidali G. Allfrey V.G. J. Biol. Chem. 1974; 249: 667-677Abstract Full Text PDF PubMed Google Scholar). Chromatin immunoprecipitation was done using a modification of the procedure of Kuo and Allis (36Kuo M.H. Allis C.D. Methods. 1999; 19: 425-433Crossref PubMed Scopus (479) Google Scholar). Cells were incubated for 15 min in medium containing 1% formaldehyde at room temperature, and cross-linking was quenched by adding glycine to 125 mm. Cells were washed with ice-cold Tris-buffered saline (150 mm NaCl, 20 mm Tris-HCl, pH 7.6) three times. Cell pellets were resuspended in 500 μl of lysis buffer (0.1% deoxycholic acid, 1 mm EDTA, 50 mm HEPES, pH 7.5, 140 mm NaCl, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin), and cells were disrupted on ice with 20 strokes of a Dounce homogenizer. Sonication was performed by pulsing three times for 15 s, incubating on ice for 2 min between each pulse, followed by centrifugation at 15,000 × g for 5 min to remove cell debris. Gel electrophoresis indicates that a substantial fraction of DNA fragments at this stage are from 0.3 to 3.0 kb in length. Centrifugation was then performed for another 15 min to obtain cleared cell lysate. ChIP was performed on the cell lysate by overnight incubation at 4 °C with 4 μg of primary antibody followed by incubation with protein G Plus-agarose (Santa Cruz Biotechnology) for 2 h. The beads were rinsed four times sequentially with lysis buffer 500 (0.1% deoxycholic acid, 1 mm EDTA, 50 mm HEPES, pH 7.5, 500 mm NaCl, 1% Triton X-100), LiCl/detergent solution (0.5% deoxycholic acid, 1 mm EDTA, 250 mm LiCl, 0.5% Non-idet P-40, 10 mm Tris-HCl, pH 8.0), and TE buffer (10 mm Tris-HCl, pH 8.0, 1 mm EDTA, pH 8.0). The beads were then incubated for 10 min at 65 °C with elution buffer (10 mm EDTA, 1% SDS, 50 mm Tris-HCl, pH 8.0) to elute the precipitates. For re-ChIP, re-ChIP elution buffer (10 mm EDTA, 50 mm Tris-HCl, pH 8.0, 0.7 m NaCl) was used to elute. The elution buffer was diluted to 1/5, and re-ChIP was performed using a second primary antibody as for the first ChIP. To reverse the cross-linking and purify the DNA, precipitates were incubated in a 65 °C incubator overnight and then incubated with proteinase K solution (40 μg/ml glycogen and 0.4 mg/ml proteinase K stock containing 50 mm Tris-HCl, pH 8.0, and 1 mm CaCl2 in TE buffer, pH 7.6) for 2 h at 37 °C. DNA samples were then purified using LiCl and phenol/chloroform/isoamyl alcohol. DNA was precipitated by adding ethanol to 70%, and precipitates were washed with 75% ethanol, air-dried, and resuspended in TE buffer. PCR Amplification of DNA Sequences Obtained by ChIP—-The following primers were employed to amplify genomic sequences upstream of the human c-MYC gene. Locations of the primers are specified by distances from the HindIII site at 2325 nt upstream of exon I specified by P1 promoter. myc5F 198, 5′-AAGCTGAATTGTGCAGTGCATC-3′ (528–549, 22-mer); myc3R 198, 5′-CTCACCCAAAGGCATTTTAAG-3′ (725–705, 21-mer); myc5F 350, 5′-CCTCTTCTTTGATCAGAATCG-3′ (1052–1072, 21-mer); myc3R 350, 5′-CCAATTTCTCAGCCAGGTTTC-3′ (1401–1381, 21-mer); myc5F segment a, 5′-CCTCCAGTAACTCCTCTTTC-3′ (175–195, 20-mer); myc3R segment a, 5′-CTCTACTGGCAGCAGAGATC-3′ (373–354, 20-mer); myc5F segment b, 5′-CATGGTGAACCAGAGTTTCATC-3′ (4845–4866, 22-mer); myc3R segment b, 5′-CTCAGATCCTGCAGGTACAAG-3′ (5063–5043, 21-mer); myc5F segment c, 5′-CAAAAGGACCATAGCAGAGAC-3′ (-3780–3760, 21-mer); myc3R segment c, 5′-CATACCTCCAGCTTGGCCTTC-3′ (-3598–3578, 21-mer); myc5F segment d, 5′-GTTGCCTCATCTATCTTGAGTG-3′ (62426–62447, 22-mer); myc3R segment d, 5′-GAGAACTCCTCCTTTCCAGTG-3′ (62605–62625, 21-mer); myc5F segment e, 5′-CGTTGTCATTTCCTATGTCCC-3′ (-61351–61331, 21-mer); and myc3R segment e, 5′-GTCCTTCTTATGTTCCAGGCAC-3′ (-61170–61149, 22-mer). PCRs were performed as previously described (37Johnson E.M. Kinoshita Y. Daniel D.C. Nucleic Acids Res. 2003; 31: 2915-2925Crossref PubMed Scopus (51) Google Scholar). Reactions were carried out with 0.2 mm of each dNTP, 0.3 μm primer/each, 10× PCR buffer with Mg2+, and 1.75 unit of Enzyme mix of the High Fidelity PCR System (Roche Applied Science). Hot-start PCR reactions were carried out for either 34 or 42 cycles as indicated. PCR reactions were analyzed by electrophoresis on 2% agarose gels stained with SYBR Gold nucleic acid gel stain (Molecular Probes). Immunoblotting, Immunoprecipitation, and Detection of Proteins— Total HeLa cell lysates were produced by Dounce homogenization in radioimmune precipitation assay buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin, in PBS (137 mm NaCl, 2.7 mm potassium chloride, 4.2 mm sodium phosphate, and 1.5 mm potassium phosphate)). Cell lysates were cleared by centrifugation. SDS-PAGE and immunoblot transfers of proteins to Immobilon-P membranes were performed as described previously (38Daniel D.C. Wortman M.J. Schiller R.J. Liu H. Gan L. Mellen J.S. Chang C.F. Gallia G.L. Rappaport J. Khalili K. Johnson E.M. J. Gen. Virol. 2001; 82: 1543-1553Crossref PubMed Scopus (44) Google Scholar, 39Barr S.M. Johnson E.M. J. Cell. Biochem. 2001; 81: 621-638Crossref PubMed Scopus (46) Google Scholar). The Supersignal chemiluminescence system (Pierce) was used for detection. Antibodies for MCM2 (N-19 goat polyclonal), MCM3 (N-19 goat polyclonal), MCM4 (K-18 goat polyclonal), MCM6 (C-20 goat polyclonal), MCM7 (141.2 mouse monoclonal), and ORC1 (N-17, R-15 goat polyclonal) were all obtained from Santa Cruz Biotechnology. Anti-Cdc6 (Ab-3 mouse monoclonal) was obtained from Oncogene. Immunoprecipitation of MCM proteins from HeLa cell lysates was performed as described previously (37Johnson E.M. Kinoshita Y. Daniel D.C. Nucleic Acids Res. 2003; 31: 2915-2925Crossref PubMed Scopus (51) Google Scholar). Immunohistochemistry of MCM7 in Tissue Sections—Paraffin sections were deparaffinized in xylene and rehydrated in 100% ethanol, 90% ethanol, and water. To quench endogenous peroxidase activity, slides were incubated for 20 min with 3% hydrogen peroxide. Antigen retrieval was performed by heating the sections in 0.1 m citrate buffer, pH 6.0, for 3 min. Sections were briefly rinsed with PBS. Slides were then incubated with primary antibody, mouse monoclonal anti-MCM7, in blocking solution (milk and goat serum in PBS) overnight at room temperature. Prior to addition of biotinylated secondary antibody and streptavidin peroxidase (BioGenex) complex for 5 min at 37 °C, sections were rinsed in PBS three times and blocked in milk/PBS. Sections were then rinsed in 0.5% Triton X-100/PBS for 10 min. The peroxidase activity was visualized using diaminobenzidine tetrahydrochloride (DAB kit, Vector). Slides were counterstained with hematoxylin, dehydrated, and mounted. Timing of Specific Localization of MCM7 in c-MYC Chromatin During the HeLa Cell Cycle—We sought a method by which we could compare simultaneous localization of a given protein at multiple points over an extended genomic sequence region. Real-time PCR was not considered practical here, because, although it provides excellent quantitation of sequence representation in a chromatin sample obtained by ChIP, the method becomes cumbersome when applied to multiple sequence locations in multiple cell cycle time points. The high degree of synchrony achieved by the double thymidine block method is shown in Fig. 1A. The adaptation of chromatin immunoprecipitation (ChIP) detailed in Fig. 1B allows assessment of the presence of a given MCM protein at two locations upstream of the c-MYC gene and, simultaneously, on an extended region between the two locations throughout the cell cycle. Fig. 1C shows the positions of two primer sets used to amplify the three indicated PCR segments. The 198-bp segment is near the center of the replication initiation zone originally mapped upstream of c-MYC (29Vassilev L. Johnson E.M. Mol. Cell. Biol. 1990; 10: 4899-4904Crossref PubMed Scopus (128) Google Scholar). It contains a prominent site of DNA bending and a purine-rich recognition element for the single-stranded DNA-binding protein, Purα (40Bergemann A.D. Johnson E.M. Mol. Cell. Biol. 1992; 12: 1257-1265Crossref PubMed Scopus (159) Google Scholar, 41Bergemann A.D. Ma Z.-W. Johnson E.M. Mol. Cell. Biol. 1992; 12: 5673-5682Crossref PubMed Scopus (134) Google Scholar). The 350-bp segment is located nearer to the c-MYC transcription start site and contains various promoter elements. The sequence between these two segments contains an extensive A-T-rich region with multiple potential DNA-unwinding sites and an element for binding another single-stranded DNA-binding protein, FBP (42Duncan R. Bazar L. Michelotti G. Tomonaga T. Krutzsch H. Avigan M. Levens D. Genes Dev. 1994; 8: 465-480Crossref PubMed Scopus (285) Google Scholar, 43Bazar L. Meighen D. Harris V. Duncan R. Levens D. Avigan M. J. Biol. Chem. 1995; 270: 8241-8248Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The 876-bp segment includes the other two segments. It should be noted, however, that due to the presence of annealed primers in the amplifying region, the 876-bp segment is not amplified as efficiently as either the 198 or 350-bp segments. We hypothesized that, because the 198-bp segment is very near the center of the reported zone of initiation, proteins known to be involved in formation of the pre-initiation complex could be located there after commitment to replication, in late G1, and prior to the onset of replication, in S-phase. The cell cycle kinetics of the double thymidine block-synchronized HeLa cells has been thoroughly documented in previous publications (34Karn J. Johnson E.M. Vidali G. Allfrey V.G. J. Biol. Chem. 1974; 249: 667-677Abstract Full Text PDF PubMed Google Scholar). We initially focused on MCM7, because it is an essential initiation component in yeast and a component of a potential helicase complex in mammalian cells. It can be seen in Fig. 1B that the presence of MCM7 differs among the three indicated PCR segments during the cell cycle. Anti-MCM7 highly selectively precipitates sequences from the 198-bp segment at 2 h (early S-phase) and 16 h (mid-to-late G1-phase) after release from the double thymidine block. At all time points tested MCM7 can be detected on this 198-bp segment. The peak of mitosis in the cell cycle of Fig. 1B occurs at ∼11.5 h. Mitosis, however, occurs very quickly relative to the scale at which time points are harvested (34Karn J. Johnson E.M. Vidali G. Allfrey V.G. J. Biol. Chem. 1974; 249: 667-677Abstract Full Text PDF PubMed Google Scholar). Therefore, the presence of MCM7 on the 198-bp segment at the 10- and 12-h points may reflect the presence of cells both pre- and post-mitosis. The detection of the 350- and 876-bp segments at 12 and 14 h suggests that some MCM7 may be present after mitosis, although the presence of a small percentage of G2 cells may contribute to this. Data to be presented later in this report indicate that there is actually a short window of time at mitosis when MCM7 is not present on the chromatin. Several aspects of the data in Fig. 1B require comment. The 0-time point was taken immediately after removal of thymidine and, thus, represents the status of the cells during blockage. Cells taken in the presence of thymidine yield similar results. At 2 and 16 h after release, when localization of MCM7 on chromatin is highly specific for the 198-bp segment, there is only slight presence of the 876-bp band. Because the larger segment includes the sequence of the smaller segment, one might expect the larger segment to be represented in the PCR products at least equally to that of the smaller one. Its lower representation reflects in part the fact that the 876-bp segment is less effectively amplified than are the two smaller segments due to steric hindrance from primers for the smaller segments. Clearly these data rely on certain controls that would attest to the quantitation of the immunoprecipitation of DNA sequences and the PCR method used to detect them. Certain of these controls, presented at the right in Fig. 1B, show that no chromatin segments are precipitated with beads alone (B), from lysate with no additions (L), or with irrelevant antibodies (a-GFP, a-GST). Additional controls are required to rule out the possibility that the size of the PCR-amplified segments could affect their amplification efficiency due to the DNA shearing during preparation. These controls are presented later in this report. The data of Fig. 1B indicate a dynamically changing distribution of MCM7 during the cell cycle, beginning with a selective localization in late G1 and early S. Note that even as MCM7 distributes to distant sites in late S and G2, it remains at its initial site on the 198-bp segment. Details of this observation will be considered under “Discussion.” ChIP of DNA Segments at Different Distances from the Initiation Zone Reveal a Dissemination of MCM7 throughout S- and G2-phases—If the presence of MCM7 is expanding outward from an initial assembly point during ongoing replication, then PCR segments that are the same size as the initial segment, but distally located, should yield results similar to those of the 350- and 876-bp segments when analyzed over the cell cycle, and the appearance of MCM7 in the more proximal segments should occur earlier than in the more distal segments. Fig. 2 shows that there is a dissemination of MCM7 to distal chromatin sites, but that dissemination is not a simple progression from a central point. Five segments have been used (a–e), all of which are at different sites from the segments used for Fig. 1. The positions of these segments are indicated in the two maps at the bottom of Fig. 2. It appears that there is increasing distribution to the segments a–d during S and G2, similar to those seen with the 350- and 876-bp segments of Fig. 1. MCM7 appears, however, in segments b and d earlier than it does in segment a, which is much closer to the 198-bp site of initial appearance. The segments closest to the initiation zone center, a–c, show the sharpest peaks of MCM7 location, from 6 to 10 h. Segments b and c are each about 4 kb from the 198-bp segment of Fig. 1, and their patterns are quite similar to that of the 350-bp segment of Fig. 1. This similar peaking may be anticipated if there is outward progression from a central segment, but the timing of MCM7 appearance is much slower than expected. Mammalian replication forks are believed to progress at >100 nt/s. Aspects of the rates of distribution of MCM7 to these segments will be considered in the discussion. Segment e shows a pattern similar to that of the 198-bp segment in Fig. 1. This segment is nearly 60 kb away from the center of the 198-bp segment and may be in or near another replicon. The patterns observed with segments a–d indicate that there is dissemination of MCM7 to multiple chromatin sites during DNA replication. If this dissemination involves progression, however, that must be coupled with an alternate, and slower, form of recruitment. Double ChIP Reveals That MCM7 and Cdc6 Are Present Together on the 198-bp c-MYC DNA Segment Specifically at Cell Cycle Times of Initiation of Replication—The notion that MCM7 is specifically localized on DNA at initiat" @default.
• W1991658906 created "2016-06-24" @default.
• W1991658906 creator A5042577601 @default.
• W1991658906 creator A5080512141 @default.
• W1991658906 date "2004-08-01" @default.
• W1991658906 modified "2023-10-11" @default.
• W1991658906 title "Site-specific Loading of an MCM Protein Complex in a DNA Replication Initiation Zone Upstream of the c-MYC Gene in the HeLa Cell Cycle" @default.
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