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• W1996739296 abstract "Mcm2, a member of the Mcm2–7 protein family essential for the initiation of DNA replication, has several biochemical activities including the ability to inhibit the Mcm4,6,7 helicase. In this study, we characterized the activities associated with Mcm2 and determined the region required for them. It was found that Mcm2 deleted at an amino-terminal portion is able to bind to an Mcm4,6,7 hexameric complex and to inhibit its DNA helicase activity. The same deletion mutant of Mcm2 and the carboxyl-terminal half of Mcm2 were both able to bind to Mcm4, suggesting that the carboxyl-half of Mcm2 binds to Mcm4 to disassemble the Mcm4,6,7 hexamer. Phosphorylation of Mcm2,4,6,7 complexes with Cdc7 kinase showed that the amino-terminal region of Mcm2 is required for the phosphorylation, and it contains major Cdc7-mediated phosphorylation sites. We also found that Mcm2 itself can assemble a nucleosome-like structure in vitro in the presence of H3/H4 histones. The amino-terminal region of Mcm2 was required for the activity where a histone-binding domain is located. Finally, we identified a region required for the nuclear localization of Mcm2. The function of Mcm2 is discussed based on these biochemical characteristics. Mcm2, a member of the Mcm2–7 protein family essential for the initiation of DNA replication, has several biochemical activities including the ability to inhibit the Mcm4,6,7 helicase. In this study, we characterized the activities associated with Mcm2 and determined the region required for them. It was found that Mcm2 deleted at an amino-terminal portion is able to bind to an Mcm4,6,7 hexameric complex and to inhibit its DNA helicase activity. The same deletion mutant of Mcm2 and the carboxyl-terminal half of Mcm2 were both able to bind to Mcm4, suggesting that the carboxyl-half of Mcm2 binds to Mcm4 to disassemble the Mcm4,6,7 hexamer. Phosphorylation of Mcm2,4,6,7 complexes with Cdc7 kinase showed that the amino-terminal region of Mcm2 is required for the phosphorylation, and it contains major Cdc7-mediated phosphorylation sites. We also found that Mcm2 itself can assemble a nucleosome-like structure in vitro in the presence of H3/H4 histones. The amino-terminal region of Mcm2 was required for the activity where a histone-binding domain is located. Finally, we identified a region required for the nuclear localization of Mcm2. The function of Mcm2 is discussed based on these biochemical characteristics. activator of S phase kinase amino acid green fluorescent protein nuclear-targeting sequence All the Mcm2–7 proteins play an essential and distinct role in eukaryotic DNA replication (1Kearsey S.E. Labib K. Biochim. Biophys. Acta. 1998; 1398: 113-136Crossref PubMed Scopus (230) Google Scholar, 2Tye B.K. Annu. Rev. Biochem. 1999; 68: 649-686Crossref PubMed Scopus (515) Google Scholar, 3Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (334) Google Scholar). They bind to chromatin during late mitosis and the G1 phase and detach from chromatin as DNA replication proceeds. It is suggested that Mcm2–7 proteins bind as a heterohexamer (4Kimura H. Ohtomo T. Yamaguchi M. Ishii A. Sugimoto K. Genes Cells. 1996; 1: 977-993Crossref PubMed Scopus (63) Google Scholar, 5Fujita M. Kiyono T. Hayashi Y. Ishibashi M. J. Biol. Chem. 1997; 272: 10928-10935Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 6Richter A. Knippers R. Eur. J. Biochem. 1997; 247: 136-141Crossref PubMed Scopus (24) Google Scholar) with the assistance of Cdc6 and Cdt1 (7Maiorano D. Moreau J. Mechali M. Nature. 2000; 404: 622-628Crossref PubMed Scopus (296) Google Scholar, 8Nishitani H. Lygerou Z. Nishimoto T. Nurse P. Nature. 2000; 404: 625-628Crossref PubMed Scopus (372) Google Scholar, 9Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (390) Google Scholar) to the region where origin recognition complex binds. In the heterohexamer, Mcm4,6,7 proteins form a stable core complex (4Kimura H. Ohtomo T. Yamaguchi M. Ishii A. Sugimoto K. Genes Cells. 1996; 1: 977-993Crossref PubMed Scopus (63) Google Scholar,10Ishimi Y. Ichinose S. Omori A. Sato K. Kimura H. J. Biol. Chem. 1996; 271: 24115-24122Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 11Sherman D.A. Pasion S.G. Forsburg S.L. Mol. Biol. Cell. 1998; 9: 1833-1845Crossref PubMed Scopus (47) Google Scholar, 12Thommes P. Kubota Y. Takisawa H. Blow J.J. EMBO J. 1997; 16: 3312-3319Crossref PubMed Scopus (117) Google Scholar, 13Lee J.-K. Hurwitz J. J. Biol. Chem. 2000; 275: 18871-18878Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), and Mcm2 has an affinity to the complex to form a tetramer of Mcm2,4,6,7. An Mcm3/5 dimer is the form most loosely associated with other Mcm proteins in the heterohexamer. Only the heterohexameric Mcm2–7 complex among these complexes has an ability to induce DNA replication in the Xenopus egg system (12Thommes P. Kubota Y. Takisawa H. Blow J.J. EMBO J. 1997; 16: 3312-3319Crossref PubMed Scopus (117) Google Scholar). The Mcm2–7 all have a DNA-dependent ATPase motif in the central domain (14Koonin E.V. Nucleic Acids Res. 1993; 21: 2541-2547Crossref PubMed Scopus (341) Google Scholar). Partly consistent with this notion, it was found that DNA helicase activity is associated with an Mcm4,6,7 hexameric complex (a dimer of Mcm4,6,7 trimer) prepared fromSchizosaccharomyces pombe (13Lee J.-K. Hurwitz J. J. Biol. Chem. 2000; 275: 18871-18878Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), mouse (15You Z. Komamura Y. Ishimi Y. Mol. Cell. Biol. 1999; 19: 8003-8015Crossref PubMed Scopus (171) Google Scholar), and human (16Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar) Mcm proteins. Mcm2 and Mcm3/5 can inhibit the DNA helicase activity by disassembling the Mcm4,6,7 hexamer into an Mcm2,4,6,7 complex or an Mcm3,4,5,6,7 complex, respectively (13Lee J.-K. Hurwitz J. J. Biol. Chem. 2000; 275: 18871-18878Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 17Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1998; 273: 8369-8375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 18Sato M. Gotow T. You Z. Komamura-Kohno Y. Uchiyama Y. Yabuta N. Nojima H. Ishimi Y. J. Mol. Biol. 2000; 300: 421-431Crossref PubMed Scopus (73) Google Scholar). These results indicate that the assembly of Mcm4,6,7 proteins into a hexamer is crucial for the DNA helicase activity and also suggest that Mcm2, -3, and -5 proteins play a role in regulating the Mcm4,6,7 helicase activity. A Cdc7/Dbf4 kinase that plays an essential role in eukaryotic DNA replication (reviewed in Ref. 19Johnston L.H. Masai H. Sugino A. Trends Cell Biol. 1999; 9: 249-252Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 20Masai H. Sato N. Takeda T. Arai K. Front Biosci. 1999; 4: 834-840Crossref PubMed Google Scholar, 21Sclafani R.A. J. Cell Sci. 2000; 113: 2111-2117Crossref PubMed Google Scholar, 22Jares P. Donaldson A. Blow J.J. EMBO Rep. 2000; 1: 319-322Crossref PubMed Scopus (74) Google Scholar) is required for the initiation of DNA replication at each replication origin (23Bousset K. Diffley J.F. Genes Dev. 1998; 12: 480-490Crossref PubMed Scopus (239) Google Scholar, 24Donaldson A.D. Fangman W.L. Brewer B.J. Genes Dev. 1998; 12: 491-501Crossref PubMed Scopus (182) Google Scholar). The substrates of the kinase remain to be determined, but genetic and biochemical evidence suggests that Mcm2 protein is one of the most important (25Lei M. Kawasaki Y. Young M.R. Kihara M. Sugino A. Tye B.K. Genes Dev. 1997; 11: 3365-3374Crossref PubMed Scopus (249) Google Scholar, 26Sato N. Arai K. Masai H. EMBO J. 1997; 16: 4340-4351Crossref PubMed Scopus (121) Google Scholar, 27Brown G.W. Kelly T.J. J. Biol. Chem. 1998; 273: 22083-22090Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 28Kumagai H. Sato N. Yamada M. Mahony D. Seghezzi W. Lees E. Arai K. Masai H. Mol. Cell. Biol. 1999; 19: 5083-5095Crossref PubMed Scopus (111) Google Scholar, 29Takeda T. Ogino K. Matsui E. Cho M.K. Kumagai H. Miyake T. Arai K. Masai H. Mol. Cell. Biol. 1999; 19: 5535-5547Crossref PubMed Scopus (94) Google Scholar, 30Oshiro G. Owens J.C. Shellman Y. Sclafani R.A. Li J.J. Mol. Cell. Biol. 1999; 19: 4888-4896Crossref PubMed Scopus (101) Google Scholar, 31Jiang W. McDonald D. Hope T.J. Hunter T. EMBO J. 1999; 18: 5703-5713Crossref PubMed Scopus (171) Google Scholar, 32Roberts B.T. Ying C.Y. Gautier J. Maller J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2800-2804Crossref PubMed Scopus (38) Google Scholar, 33Weinreich M. Stillman B. EMBO J. 1999; 18: 5334-5346Crossref PubMed Scopus (227) Google Scholar, 34Jares P. Blow J.J. Genes Dev. 2000; 14: 1528-1540PubMed Google Scholar, 35Kihara M. Nakai W. Asano S. Suzuki A. Kitada K. Kawasaki Y. Johnston L.H. Sugino A. J. Biol. Chem. 2000; 275: 35051-35062Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 36Takeda Ogino K. Tatebayashi K. Ikeda H. Arai K. Masai M. Mol. Biol. Cell. 2001; 12: 1257-1274Crossref PubMed Scopus (89) Google Scholar). We reported that Mcm2 can inhibit the DNA helicase activity of the Mcm4,6,7 complex (17Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1998; 273: 8369-8375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In the present study, we further analyzed the interplay between Mcm2 and the Mcm4,6,7 helicase and also determined the region in Mcm2 that is required for in vitrophosphorylation with a human Cdc7/ASK1 kinase. In addition, we found that Mcm2 can assemble nucleosome-like structuresin vitro, and the activity is related with the histone binding ability of Mcm2. Human Mcm4,6,7 hexameric complex was purified from HeLa cells by histone-Sepharose column chromatography and then by glycerol gradient centrifugation as reported (16Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). For preparing deletion mutants of mouse Mcm2 protein, theMcm2 gene was truncated by polymerase chain reaction using the following primers; as a forward primer, 5′-GAGAGAGAATTCGATCCCCTCACCTCCAGCCCAGGC-3′ (nucleotides from the mouse Mcm2 gene are underlined) for starting at amino acid (aa) 22, 5′-GAGAGAGAATTCGACCTCCCCCCATTTGAAGATGAG-3′ for starting at aa 45, 5′-GAGAGAGAATTCATGGAGGAAGAAGAGGATGG-3′ for starting at aa 63, 5′-TCGAATTCATGCTGGATGATGAAGATGTGGAGGAG-3′ for starting at aa 97, 5′- GACGAATTCGAGGATGAAGAGATGATCGAGAGTA-3′ for starting at aa 163, and 5′-GAGAGAGAATTCATGAACAAAGTAGCTGTGGGGGAGCTC -3′ for starting at aa 449. As a reverse primer, 5′-TCGAATTCTTAATGGATGTGGTTGGTGATACGG-3′ for ending at aa 282, and 5′-GAGTGAGCGGCCGCTCAGTCCTTCTTGGCAACA TGGTTGGC-3′ for ending at aa 448 were used. These Mcm2 genes were cloned in a pAcHLT-A vector (Pharmingen) for production of histidine-tagged Mcm2 proteins in a baculovirus expression system. When mouse Mcm2,4,6,7 complexes were prepared, High5 cells were co-infected with three vi- ruses for producing the histidine-tagged Mcm2, histidine-tagged Mcm4 and Mcm6 (pAcUW31vector) (15You Z. Komamura Y. Ishimi Y. Mol. Cell. Biol. 1999; 19: 8003-8015Crossref PubMed Scopus (171) Google Scholar), and Mcm7 proteins (pVL1392 vec- tor). The Mcm2 proteins and Mcm2,4,6,7 complexes were purified by Ni2+ column chromatography, and the Mcm2,4,6,7 complexes were fur- ther purified by glycerol gradient centrifugation as reported. A human glutathioneS-transferase-huCdc7/myc-ASK complex was purified from High5 cells co-infected with the recombinant baculoviruses (37Masai H. Matsui E. You Z. Ishimi Y. Tamai K. Arai K. J. Biol. Chem. 2000; 275: 29042-29052Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), and the complex was purified by glutathione-Sepharose chromatography as the manufacturer suggested (Pharmingen). Briefly, cell lysate was mixed with a 1/10 volume of glutathione agarose and incubated for 30 min at 4 °C. Then the agarose was washed with phosphate-buffered saline, and the human glutathione S-transferase-Cdc7/myc-ASK complex was eluted with 5 mm glutathione in 50 mmTris-HCl, pH 8.0. The Mcm2,4,6,7 complexes (40–400 ng) were incubated with various amounts of the huCdc7/ASK complex at 30 °C for 30 min in a 20-μl reaction mixture consisting of 40 mm Hepes-KOH, pH 8.0, 40 mm potassium glutamate, 1 mm EGTA, 8 mm magnesium acetate, 2 mm dithiothreitol, 0.5 mm EDTA, 0.1 mm ATP, and 0.01% Triton X-100 in the presence of [γ-32P]ATP. The phosphorylated proteins were analyzed in 10% polyacrylamide gel containing SDS. The same sample was digested with lysyl endopeptidase in the presence of 0.1% SDS and analyzed in 15–25% polyacrylamide gel. Each of the mouse Mcm2 and Mcm6 genes was cloned in pBluescript II SK plasmid (Stratagene). Truncated forms of theMcm2 gene were amplified by polymerase chain reaction using primers from each site. An ATG sequence was added to the 5′-end of the forward primers to create a methionine at the amino terminus of the Mcm2 proteins. Site-directed mutagenesis of the Mcm2 gene was conducted using the QuikChange site-directed mutagenesis kit (Stratagene). The oligonucleotide 5′- GTCAAGTACAACGCTAGCAAGGCCAACTTTGTACTGGGGC-3′ was used as a primer to introduce changes from Cys to Ala at positions 329 and 332 in a putative zinc finger motif. The resulting DNAs were cloned into pBluescript II SK. The mutated sites in Mcm2 were confirmed by nucleotide sequencing. Various Mcm2 proteins and native Mcm6 protein were synthesized in vitro in the presence of [35S]methionine in a reticulocyte lysate system, as suggested by the manufacturer (TNT- coupled reticulocyte lysate system, Promega). Histidine-tagged mouse Mcm4 proteins were prepared by Ni2+ column chromatography after production in insect cells, and they were fixed with CNBr-activated Sepharose at the concentration of 0.5 mg/ml. Bovine serum albumin fixed with the Sepharose was prepared as control beads. Aliquots of the [35S]methionine-labeled Mcm proteins were mixed with the mouse Mcm4-beads for 1 h at 4 °C, and the beads were washed five times with phosphate-buffered saline containing 0.05% Nonidet P-40. Proteins bound to the beads were eluted with SDS and electrophoresed on polyacrylamide gel. Site-directed mutagenesis of the Mcm2 gene was conducted. The oligonucleotide 5′-CTCCAGCCCAGGCCTTAGCTCCATACTTGCTGACGCCCTG-3′ was used as a primer to introduce changes from Arg to Leu at position 30, from Arg to Ile at position 33, and from Arg to Leu at position 34 in NTS1 (18–34), whereas 5′-GAAGATGAGGAGCTGCCTGCCCTTGCGCGCCGCCACGTAG-3′ was used for changes from Arg to Leu at position 146, from Arg to Leu at position 149, and Lys to Ala at position 150 in NTS2 (132). The native form as well as the mutagenized forms of the mouseMcm2 gene were cloned into pEGFP-N1 (CLONTECH) to synthesize Mcm2-GFP fusion proteins where the carboxyl-terminal end of the Mcm2 protein was fused to the amino-terminal end of GFP. The cloned DNAs were transfected into mouse L cells using a CalPhos Maximizer transfection kit (CLONTECH). The cells (5 × 104) were grown in 8-well chambers with 250 μl of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum for 1 day. The cloned DNA (1.5 μg) was mixed with CalPhos Maximizer, calcium solution, and Hepes-buffered saline and left for 15 min at room temperature. The DNA solution was added to the cells in culture medium. The dish was placed at 37 °C in a CO2incubator for 5 h. The DNA solution was removed, growth medium was added, and the dish was returned to the incubator. After 2 days, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 30 min at room temperature. After three washes with phosphate-buffered saline, the fluorescence in the cells was observed by Olympus AX80 and photographs were taken. A 17-mer oligonucleotide (5′-GTTTTCCCAGTCACGAC-3′) was labeled at the 5′-end with polynucleotide kinase in the presence of [γ-32P]ATP and then annealed to M13 DNA. The annealed oligomer (2.5–5 fmol) was incubated at 37 °C for 30 min with an Mcm4,6,7 complex in the presence or absence of Mcm2 protein in 50 mm Tris-HCl, pH 7.9, 20 mm 2-mercaptoethanol, 10 mm magnesium acetate, 10 mm ATP, and 0.5 mg/ml bovine serum albumin. The reaction was terminated by adding 0.2% SDS, and an aliquot was electrophoresed on a 12% acrylamide gel in Tris borate/EDTA. The labeled oligomer in the gel was detected by using a Bio-Image Analyzer (FLA2000, Fuji). To analyze the effect of Mcm2 on the complex formation of Mcm4,6,7 proteins, increasing amounts of Mcm2 proteins were incubated with an Mcm4,6,7 complex for 30 min at 37 °C in 50 mm Tris-HCl, pH 7.9, 20 mm2-mercaptoethanol, 5 mm ATP, 5 mmMgCl2, and 0.01% Triton X-100, and the mixture was analyzed under non-denaturing conditions by native 5% acrylamide gel electrophoresis. The proteins in the gel were transferred to a nitrocellulose membrane after incubation in 49 mm Tris-HCl, pH 6.8, 38 mm glycine, and 0.25% SDS at 80 °C for 1 h, and Mcm4 on the membrane was detected by using anti-Mcm4 antibodies. Plasmid DNA (pSV01EP, 100 ng) was incubated with topoisomerase I in 10 mmcreatine-phosphate (sodium salt), pH 7.8, 4 mm ATP, 7 mm MgCl2, 0.4 mm dithiothreitol, 25 μg/ml creatine phosphokinase, 400 μg/ml bovine serum albumin, and 20 mm potassium phosphate for 15 min at 37 °C. Indicated amounts of Mcm2 protein were mixed with H3/H4 histones (300 ng) in the same buffer as described above for 15 min. These two solutions were combined, and the reaction was further run for 45 min. After purification, DNA was electrophoresed in 1% agarose gel and stained with ethidium bromide. We reported that Mcm2 can inhibit the DNA helicase activity of an Mcm4,6,7 hexamer by disassembling the hexamer into an Mcm2,4,6,7 tetramer (17Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1998; 273: 8369-8375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Mcm4,6,7 proteins mainly formed a hexameric complex or else a trimeric complex, as was determined by native acrylamide gel electrophoresis followed by immunoblotting using anti-Mcm4 antibodies (Fig.1 A). In the presence of full-size mouse Mcm2, these mouse Mcm4,6,7 complexes were converted to a tetrameric Mcm2,4,6,7 complex. We examined the region in Mcm2 that is required for the disassembly of the Mcm4,6,7 hexamer into the Mcm2,4,6,7 complex. The Mcm4,6,7 complex was incubated with mutant Mcm2 proteins, and then the changes in molecular mass of Mcm complexes were examined. Similarly to full-size Mcm2, a mutant Mcm2 that is deleted at the amino-terminal region (1) retained the activity to disassemble the Mcm4,6,7 hexamer and to form the Mcm2,4,6,7 tetramer (Fig.1 A). In the presence of larger amounts of the mutant Mcm2 protein, the mobility of the Mcm2,4,6,7 complex was retarded in native gel, suggesting that more than one molecule of Mcm2 protein is bound to the Mcm4,6,7 trimer under these conditions. The formation of the Mcm2,4,6,7 complex was observed in the presence of non-hydrolyzable ATP analogues in place of ATP in the reaction mixture (data not shown). In contrast, the amino-terminal half (1) of the Mcm2 protein did not show any disassembly activity (Fig. 1 B). Next, the effect of mutant Mcm2 proteins on the DNA helicase activity of the Mcm4,6,7 hexamer was examined (Fig. 2). Mcm2 deleted at the amino-terminal end (1) showed inhibition of DNA helicase activity similar to the full-sized Mcm2 protein (17Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1998; 273: 8369-8375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). However, an amino-terminal fragment (1) of Mcm2 did not show inhibitory activity. These results indicate that the amino-terminal region of Mcm2 is dispensable not only for disassembly of the Mcm4,6,7 hexamer but also for the inhibition of the DNA helicase activity of the Mcm4,6,7 hexamer.Figure 2Inhibition of Mcm4,6,7 helicase activity by Mcm2. A, Mcm4,6,7 complex (100 ng) was examined for DNA helicase activity in the presence of two different amounts (25 and 50 ng) of Mcm2 mutants (Δ1–162, 1–282). The position of the released 17-mer is indicated. At the bottom (B), a summary of the results is shown.View Large Image Figure ViewerDownload (PPT) From pull-down experiments, we found that Mcm2 protein mainly interacts with Mcm4 among Mcm4, -6, and -7 proteins. 2Y. Ishimi, unpublished results. To determine the region in Mcm2 that is required for the interaction with Mcm4, we prepared three deletion mutants of Mcm2 protein. The mutant Mcm2 deleted at the amino-terminal region (1) and the amino-half (1) were detected as several bands in SDS-gel, which may be alternatives of the in vitro translation products. These proteins and also Mcm6 protein, as a control, were mixed and incubated with mouse Mcm4-beads, and proteins bound to the beads were analyzed on SDS-polyacrylamide gel (Fig. 3). The results showed that both a mutant Mcm2 deleted at the amino-terminal region (1) and a carboxyl-half (449) of Mcm2 were capable of binding to the Mcm4-beads but not to bovine serum albumin beads (Fig.3). Approximately 7% of radioactivity was recovered in the Mcm4-bound fraction for the Mcm2 proteins of full size and Δ1–166 and 11–12% for Mcm2 (449) and for Mcm6 protein. In contrast, the amino-half (1) did not bind to Mcm4. Less than 2% of radioactivity was recovered in the bound fraction for the amino-half of Mcm2 protein. These results indicate that the carboxyl-half of Mcm2 plays an important role in interacting with Mcm4 protein, although the possibility that the binding is not due to a direct interaction of bound proteins with Mcm4 cannot be excluded. Consistent with the above notion, mutations at a putative Zn2+ finger motif in the amino-half of Mcm2 did not affect the binding of Mcm2 to the Mcm4 beads. These results and those in Figs. 1 and 2 suggest that Mcm2 protein disassembles Mcm4,6,7 hexamer through the interaction of its carboxyl-half with Mcm4 in the hexamer. A number of reports indicate that Mcm2 protein is a good substrate of Cdc7 kinase in vitro (25–37). We incubated a purified Mcm2,4,6,7 complex with the human Cdc7/ASK kinase complex (Fig.4, A and B). Consistent with the previous studies, Mcm2 of full-size was specifically phosphorylated with the kinase in the Mcm2,4,6,7 complex under these conditions. To map the region in Mcm2 that is required for the phosphorylation with the Cdc7/ASK kinase, we constructed four Mcm2 proteins deleted at the amino-terminal region and then purified Mcm2,4,6,7 complexes containing these Mcm2 (Fig. 4 A). The Mcm2 deleted at amino acids 1–44 showed an unexpected mobility in SDS-gel. We incubated these complexes with Cdc7/ASK kinase (Fig. 4,B and C). In contrast to full-size Mcm2, all the three mutant Mcm2 proteins in which the amino-terminal region was deleted to different extents (Δ1–62, Δ1–162, and Δ1–448) were hardly phosphorylated by the kinase (Fig. 4 B). To further map the region required for the phosphorylation, we prepared two additional deletion mutants of Mcm2 (Δ1–21 and Δ1–44) and phosphorylated the Mcm2,4,6,7 complexes containing them with the Cdc7 kinase (Fig. 4 C). A slight decrease in the phosphorylation was detected in the Mcm2 deleted at 1–21, and a relatively distinct decrease was detected in the Mcm2 deleted at 1–44. The results indicate that the amino-terminal region (1–62) of mouse Mcm2 plays a crucial role in the phosphorylation by Cdc7/ASK kinase (Fig.4 D). Phosphorylated full-size Mcm2 appears as single band in Fig. 4 B, whereas it appears as two bands in Fig.4 C and Fig. 5 A. Comparison of number of incorporated phosphates in these experiments suggests that a higher level of phosphorylation leads to generation of an extra band of phosphorylated Mcm2 on SDS-gel.Figure 5Quantification of Mcm2 phosphorylation. A, Mcm2,4,6,7 complex containing full-sized Mcm2 was phosphorylated with three different amounts of Cdc7/ASK, and proteins were electrophoresed in 10% polyacrylamide gel containing SDS.B, phosphorylated proteins in A were digested with lysyl endopeptidase and analyzed on 15–25% acrylamide gel.C, the number of phosphate molecules incorporated into full-size Mcm2 (horizontal line) and into the amino-terminal fragment (1) (vertical line) in the above experiments were calculated and plotted with closed circles. Several other experiments are included in the graph where the data in the same experiment were plotted with the same symbols.View Large Image Figure ViewerDownload (PPT) To address the issue of whether the amino-terminal region of Mcm2 contains major Cdc7-mediated phosphorylation sites, the phosphorylated full-sized Mcm2 in Mcm2,4,6,7 complex was digested with lysyl endopeptidase to analyze the phosphorylation in the amino-terminal region of Mcm2 (Fig. 5). The digestion generated a fragment of amino acids, 1–150 of Mcm2 (see Fig. 8 B), that was detected as a 45-kDa band in SDS-gel (Fig. 5 B). Incorporation of32P into full-sized Mcm2 and into the amino-terminal fragment in the same sample was quantified in several experiments where the phosphorylation was carried out at the different levels of Cdc7 kinase; the results are shown in Fig. 5 C. When one molecule of phosphate was incorporated into full-size Mcm2, one molecule of phosphate was recovered in the amino-terminal fragment of Mcm2, indicating that Cdc7 kinase phosphorylates only the amino-terminal region. When 3 mol of phosphate were incorporated into full-size Mcm2, however, two molecules were recovered in the amino-terminal region, suggesting that one mol of phosphate is incorporated into the region other than the amino terminus. These results and those in Fig. 4 suggest that the amino-terminal region of Mcm2 plays a role in the phosphorylation in the region other than the amino-terminal region in addition to the role as the phosphorylation sites. We determined the histone binding activity in the amino-terminal region of Mcm2 (17Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1998; 273: 8369-8375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The Mcm2 in Mcm complexes specifically bound to histone H3 among four core histones. To elucidate the physiological role of the histone binding activity, we measured the nucleosome assembly activity of mouse Mcm2 protein (Fig. 6). The histone H3/H4, instead of four core histones, was used for the nucleosome assembly assay (38Bina-Stein M. Simpson R.T. Cell. 1977; 11: 609-618Abstract Full Text PDF PubMed Scopus (71) Google Scholar). If nucleosome-like structures were formed on circular DNA, negative supercoils of DNA would be generated in this system. Thus, the recovery of negatively supercoiled form-I DNA suggests that the plasmid DNA is fully assembled into nucleosome-like structures. Full-sized Mcm2 was able to stimulate the nucleosome assembly in vitro, similar to Nap-1, which showed nucleosome assembly activity in vitro (39Ishimi Y. Kojima M. Yamada M. Hanaoka F. Eur. J. Biochem. 1984; 162: 19-24Crossref Scopus (66) Google Scholar), although the level of the activity in Mcm2 was lower than that in Nap-1 (Fig. 6 A). We also determined the region in Mcm2 that is required for the activity by preparing several deletion mutants (Fig. 6, A and B). The results indicated that the deletion of 162 amino acids from the amino terminus abolished the nucleosome assembly activity (Fig. 6 A). However, the deletion of 62 amino acids from the terminus did not perturb the activity, and the removal of 96 amino acids reduced the activity (Fig.6 B). Thus, the region spanning amino acids 63–162 at the amino-terminal end of Mcm2 is required for the activity, although the amino-terminal region (1) of Mcm2 was not sufficient for the activity (Fig. 6 A). The region 63–162 almost overlapped with the histone H3 binding domain of Mcm2 identified previously. Thus, it is suggested that Mcm2 stimulates nucleosome assembly in vitro by interacting with histone H3/H4 through the histone H3 binding domain. We showed that the amino-terminal region (1) is required for the nuclear localization of mouse Mcm2 when it was expressed in HeLa cells as a GFP fusion protein (17Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1998; 273: 8369-8375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). There are two bipartite-type nuclear-targeting sequences (40Dingwall C. Laskey R.A. Trends Biol. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1713) Google Scholar) in this region of mouse Mcm2, one at position 18–34 (NTS1) and the other at 132–152 (NTS2). We introduced mutations into these two sequences and examined the effect on the nuclear localization of Mcm2-GFP fusion proteins expressed in mouse L cells (Fig.7). The mutations in NTS1 did not alter the nuclear localization of the fusion protein, but the mutations in NTS2 significantly perturbed the nuclear accumulation of the fusion protein. The Mcm2-GFP fusion protein mutated at the NTS2 was detected not only in nuclei but also in cytoplasm. When both of the NTS1 and 2 were mutated, the fusion protein was also detected both in nuclei and cytoplasm. These results suggest that the NTS2 plays an important role in the nuclear localization of Mcm2 under these conditions. This conclusion is consistent with the previous observations that the amino-terminal deletion up to amino acids 92 did not affect the nuclear localization of Mcm2 (17Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1998; 273: 8369-8375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In this paper, we analyzed the biochemical activity related to the mouse Mcm2 protein, which includes that to inhibit Mcm4,6,7 helicase and to assemble nucleosomes in vitro. We determined the region in Mcm2 required for the phosphorylation by Cdc7/ASK kinase and the region required for nuclear localization of Mcm2. We localized these activities in Mcm2, as shown in Fig.8. The results indicated that the carboxyl-half of Mcm2 is required for interacting with Mcm4 as well as for inhibiting the DNA helicase activity of Mcm4,6,7 complex. We showed that the amino-terminal region of Mcm2 where a histone H3 binding domain and a region required for nuclear localization are present is required for the phosphorylation by Cdc7 kinase. In this region, a number of Ser and Thr residues are present in addition to three consensus sites for cyclin-dependent kinase. Mcm2–7 are all essential for eukaryotic DNA replication, but only the heterohexameric Mcm2–7 complex among several Mcm complexes has the ability to induce DNA replication in the Xenopus egg system (12Thommes P. Kubota Y. Takisawa H. Blow J.J. EMBO J. 1997; 16: 3312-3319Crossref PubMed Scopus (117) Google Scholar). All the members have a DNA-dependent ATPase motif, but the heterohexameric Mcm2–7 complex shows neither significant ATPase activity or DNA helicase activity (41Adachi Y. Usukura J. Yanagida M. Genes Cells. 1997; 2: 467-479Crossref PubMed Scopus (108) Google Scholar). In contrast, an Mcm4,6,7 complex as a hexamer showed an ATP-dependent DNA helicase activity in vitro (13Lee J.-K. Hurwitz J. J. Biol. Chem. 2000; 275: 18871-18878Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 16Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). It has been proposed that a structural change in the Mcm2–7 heterohexamer or a change in protein composition occurs in the Mcm complex (17Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1998; 273: 8369-8375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 42Tye B.K. Sawyer S. J. Biol. Chem. 2000; 275: 34833-34836Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Genetically Mcm proteins interacts with Cdc7/Dbf4 in Saccharomyces cerevisiae (25Lei M. Kawasaki Y. Young M.R. Kihara M. Sugino A. Tye B.K. Genes Dev. 1997; 11: 3365-3374Crossref PubMed Scopus (249) Google Scholar, 43Hardy C.F. Dryga O. Seematter S. Pahl P.M. Sclafani R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3151-3155Crossref PubMed Scopus (218) Google Scholar). It has been shown that Mcm2 is a good substrate of the kinase in vitro (25–37). Thus, it is probable that Cdc7 kinase is involved in these changes in the Mcm complex. One possibility is that phosphorylation of Mcm2 by the kinase is involved in a structural change of the Mcm2–7 heterohexamer by which the DNA helicase activity that may intrinsically reside in the complex is induced (42Tye B.K. Sawyer S. J. Biol. Chem. 2000; 275: 34833-34836Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The notion that the heterohexameric Mcm2–7 complex acts as a DNA helicase in vivo is supported by the following findings. First, Mcm proteins appear to migrate on chromosome after the initiation of DNA replication in S. cerevisiae(44Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). Second, it has been demonstrated that all the Mcm proteins are required for elongation of DNA replication after initiation has occurred (45Labib K. Tercero J.A. Diffley J.F.X. Science. 2000; 288: 1643-1647Crossref PubMed Scopus (523) Google Scholar). In addition, it has been indicated that Mcm proteins mainly form the Mcm2–7 heterohexamer in vivo. Another possibility is that an Mcm4,6,7 complex is generated from the Mcm2–7 complex, and the phosphorylation of Mcm2 by Cdc7 kinase is involved in the removal of Mcm2,3,5 proteins (17Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1998; 273: 8369-8375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This possibility is supported by the following findings. First, only the Mcm4,6,7 hexameric complex showed DNA helicase activity among several Mcm complexes including the heterohexameric Mcm2–7 complex that were all prepared in a baculovirus expression system.2 Second, the genetic evidence that the requirement of Cdc7 kinase was bypassed by mutation of Mcm5 in S. cerevisiae suggests that Mcm5 at least functions as a negative regulator of DNA replication (43Hardy C.F. Dryga O. Seematter S. Pahl P.M. Sclafani R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3151-3155Crossref PubMed Scopus (218) Google Scholar). Consistent with this finding, both Mcm2 and Mcm3/5 complex can inhibit Mcm4,6,7 helicase activity in vitro (13Lee J.-K. Hurwitz J. J. Biol. Chem. 2000; 275: 18871-18878Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 17Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1998; 273: 8369-8375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 18Sato M. Gotow T. You Z. Komamura-Kohno Y. Uchiyama Y. Yabuta N. Nojima H. Ishimi Y. J. Mol. Biol. 2000; 300: 421-431Crossref PubMed Scopus (73) Google Scholar). Third, the Mcm4,6,7 proteins form relatively stable complexes of a monomer and a dimer of the 4,6,7 trimer, and Mcm2,3,5 proteins are relatively loosely associated with the Mcm4,6,7 trimer in the Mcm2–7 heterohexamer. This structural information appears to be consistent with the change in the composition of the Mcm complex. It is possible that the ATP binding motif of Mcm2,3,5 proteins is involved in releasing Mcm4,6,7 complex from the heterohexamer at the time when the Mcm complex functions as a DNA helicase. A similar example can be seen at the initiation of DNA replication in E. coli where a DnaC protein, which is an ATPase but an inhibitor of DnaB helicase, forms a complex with DnaB and binds to the origin region. In this system, it appears that the ATPase activity of DnaC is required for releasing DnaB at the initiation of DNA replication (46Wahle E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2469-2475Abstract Full Text PDF PubMed Google Scholar). The second model, that the Mcm4,6,7 complex generated from the Mcm2–7 complex functions as a DNA helicase in vivo, may not be inconsistent with the finding that all the Mcm2–7 proteins are required for elongation of DNA replication as well as initiation (45Labib K. Tercero J.A. Diffley J.F.X. Science. 2000; 288: 1643-1647Crossref PubMed Scopus (523) Google Scholar). Furthermore, the possibility that the Mcm4,6,7 helicase functions in DNA replication is supported by the recent finding that the Mcm4,6,7 complex from S. pombe acts as a processive DNA helicase when a tailed substrate is used for the reaction (47Lee J.-K. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 54-59Crossref PubMed Scopus (159) Google Scholar). However, we have no direct evidence for this second model. The present finding indicating that Cdc7/ASK kinase mainly phosphorylates the amino-terminal region of Mcm2, which is not required for interacting with the Mcm4,6,7 complex, seems to be inconsistent with the model. However, our results suggest that the region other than the amino-terminal region is also phosphorylated with Cdc7/ASK (Fig.5). We have another piece of data indicating that other regions of the Mcm2 which include the carboxyl-terminal segment of the protein in the Mcm2,4,6,7 complex are also phosphorylated by the kinase in vitro (data not shown). These phosphorylations at regions other than the amino terminus may modulate the structure of the Mcm complex or influence the interaction of Mcm2 with other components of the Mcm complex. Further efforts are clearly necessary to clarify the mechanism of Mcm function. It also remains to be determined to what extent Mcm helicase is responsible for replication fork movement in vivo (48Labib K. Diffley J.F.X. Curr. Opin. Genet. Dev. 2001; 11: 64-70Crossref PubMed Scopus (125) Google Scholar). Cdc7/ASK kinase phosphorylated an amino-terminal region of Mcm2 where a histone H3 binding domain and a region required for nuclear localization are present. We do not know the effect of the phosphorylation of Mcm2 by Cdc7/ASK kinase on these two activities. It has been reported that Mcm2 proteins, which are detached from chromatin during the S phase, are more phosphorylated than chromatin-bound Mcm2 proteins (49Todorov I.T. Attaran A. Kearsey S.E. J. Cell Biol. 1995; 129: 1433-1445Crossref PubMed Scopus (204) Google Scholar), suggesting that the phosphorylation of Mcm2 is involved in the detachment of Mcm proteins from chromatin. It has been shown that Cdc2/cyclinB is involved in the phosphorylation of Mcm2 proteinsin vivo using a temperature-sensitive mutant of the kinase (50Fujita M. Yamada C. Tsurumi T. Hanaoka F. Matsuzawa K. Inagaki M. J. Biol. Chem. 1998; 273: 17095-17101Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We observed that the amino-terminal region of Mcm2 is phosphorylated by Cdk2/cyclinA or Cdk2/cyclinE in vitro(data not shown). Thus, both cyclin-dependent kinase and Cdc7 kinase may be involved in the phosphorylation of the amino-terminal region of Mcm2 proteins (34Jares P. Blow J.J. Genes Dev. 2000; 14: 1528-1540PubMed Google Scholar, 37Masai H. Matsui E. You Z. Ishimi Y. Tamai K. Arai K. J. Biol. Chem. 2000; 275: 29042-29052Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In fact, prior phosphorylation of the Mcm2,4,6,7 complex with Cdk stimulated subsequent phosphorylation of Mcm2 by Cdc7 (37Masai H. Matsui E. You Z. Ishimi Y. Tamai K. Arai K. J. Biol. Chem. 2000; 275: 29042-29052Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). It has been also shown that cyclin-dependent kinase plays a role in the initiation of DNA replication by loading Cdc45 protein at the replication origin (51Zou L. Stillman B. Science. 1998; 280: 593-596Crossref PubMed Scopus (275) Google Scholar, 52Mimura S. Takisawa H. EMBO J. 1998; 17: 5699-5707Crossref PubMed Scopus (194) Google Scholar). We showed that the region around amino acid 150 (NTS2) is required for nuclear localization of mouse Mcm2. The nuclear localization of Mcm2 may depend on nuclear import of the protein and also on nuclear retention of the protein (53Young M.R. Suzuki K. Yan H. Gibson S. Tye B.K. Genes Cells. 1997; 2: 631-643Crossref PubMed Scopus (23) Google Scholar). The results in Fig. 7 suggest that mutation in the NTS2 did not affect the nuclear import but affected the retention of Mcm2 in the nucleus. In S. pombe, both of two regions of amino acids 5–10 and 114–118 are required for nuclear localization of Mcm2 (54Pasion G. Forsburg S.L. Mol. Biol. Cell. 1999; 10: 4043-4057Crossref PubMed Scopus (69) Google Scholar). The region near the amino terminus functions as a nuclear localization signal. Finally, we detected in Mcm2 activity that stimulates nucleosome assembly in vitro. The histone binding domain in Mcm2 was required for the activity. Thus, Mcm2 protein may bind with histone H3/H4 and transfer them to DNA to assemble nucleosome-like structures in vitro. The physiological significance of the activity remains to be clarified. It is possible, however, that the activity is involved in chromatin assembly during DNA replication by interacting with parental or newly synthesized histones. Conversely, it may be involved in DNA replication by disassembling nucleosomes to facilitate unwinding of DNA. Recently, it has been reported that Mcm proteins are involved in transcriptionin vivo (55Yankulov 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 (74) Google Scholar, 56DaFonseca C. Shu F. Zhang J.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3034-3039Crossref PubMed Scopus (69) Google Scholar), and a histone acetyltransferase interacts with the histone H3 binding domain of Mcm2 (57Burke T.W. Cook J.G. Asano M. Nevins J.R. J. Biol. Chem. 2001; 276: 15397-15408Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Mcm proteins may play a role in several chromatin activities in addition to having an essential role in DNA replication. We thank Etsuko Matsui for excellent technical assistance in preparing Cdc7/ASK kinase complex." @default.
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• W1996739296 title "Biochemical Activities Associated with Mouse Mcm2 Protein" @default.
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