A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex

All six minichromosome maintenance (MCM) proteins have DNA-dependent ATPase motifs in the central domain which is conserved from yeast to mammals. Our group purified MCM protein complexes consisting of MCM2, -4 (Cdc21), -6 (Mis5), and -7 (CDC47) proteins from HeLa cells by using histone-Sepharose column chromatography (Ishimi, Y., Ichinose, S., Omori, A., Sato K., and Kimura, H. (1996) J. Biol. Chem. 271, 24115–24122). The present study revealed that both ATPase activity and DNA helicase activity that displaces oligonucleotides annealed to single-stranded circular DNA are associated with an MCM protein complex. Both ATPase and DNA helicase activities were co-purified with a 600-kDa protein complex that is consisted of equal amounts of MCM4, -6, and -7 proteins. An immunodepletion of the MCM protein complex from the purified fraction using anti-MCM4 antibody resulted in the severe reduction of the DNA helicase activity. Displacement of DNA fragments by the DNA helicase suggested that it migrated along single-stranded DNA in the 3′ to 5′ direction, and the DNA helicase activity was detected only in the presence of hydrolyzable ATP or dATP. These results suggest that this helicase may be involved in the initiation of DNA replication as a DNA unwinding enzyme. All six minichromosome maintenance (MCM) proteins have DNA-dependent ATPase motifs in the central domain which is conserved from yeast to mammals. Our group purified MCM protein complexes consisting of MCM2, -4 (Cdc21), -6 (Mis5), and -7 (CDC47) proteins from HeLa cells by using histone-Sepharose column chromatography (Ishimi, Y., Ichinose, S., Omori, A., Sato K., and Kimura, H. (1996) J. Biol. Chem. 271, 24115–24122). The present study revealed that both ATPase activity and DNA helicase activity that displaces oligonucleotides annealed to single-stranded circular DNA are associated with an MCM protein complex. Both ATPase and DNA helicase activities were co-purified with a 600-kDa protein complex that is consisted of equal amounts of MCM4, -6, and -7 proteins. An immunodepletion of the MCM protein complex from the purified fraction using anti-MCM4 antibody resulted in the severe reduction of the DNA helicase activity. Displacement of DNA fragments by the DNA helicase suggested that it migrated along single-stranded DNA in the 3′ to 5′ direction, and the DNA helicase activity was detected only in the presence of hydrolyzable ATP or dATP. These results suggest that this helicase may be involved in the initiation of DNA replication as a DNA unwinding enzyme. There are at least six MCM 1The abbreviations used are: MCM, minichromosome maintenance; DTT, dithiothreitol; DSP, dithiobis-succinimidyl propionate; ATPγS, adenosine 5′-O-(thiotriphosphate); ORC, origin recognition complex; AMP-PNP, β-γ-imidoadenosine-5′-triphosphate; RLF, replication licensing factor.1The abbreviations used are: MCM, minichromosome maintenance; DTT, dithiothreitol; DSP, dithiobis-succinimidyl propionate; ATPγS, adenosine 5′-O-(thiotriphosphate); ORC, origin recognition complex; AMP-PNP, β-γ-imidoadenosine-5′-triphosphate; RLF, replication licensing factor. proteins that play an essential role in eukaryotic DNA replication as follows: MCM2, -3, -4 (Cdc21), -5, -6 (Mis5), and -7 (CDC47) proteins (see reviews in Refs.1Tye B.-K. Trends Cell Biol. 1994; 4: 160-166Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 2Chong J.P.J. Thömmes P. Blow J.J. Trends Biochem. Sci. 1996; 21: 102-106Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 3Kearsey S.E. Mariorano D. Holmes E.C. Todorov I.T. BioEssay. 1996; 18: 183-190Crossref PubMed Scopus (120) Google Scholar). Genetic evidence suggests that these proteins are required for the initiation of DNA replication in yeast (4Hennessy K.M. Lee A. Chen E. Botstein D. Genes Dev. 1991; 5: 958-969Crossref PubMed Scopus (229) Google Scholar, 5Mariorano D. Blom van Assendelft G. Kearsey S.E. EMBO J. 1996; 15: 861-872Crossref PubMed Scopus (83) Google Scholar). The inactivation of human MCM2 protein with antibody consistently inhibits the initiation of DNA replication (6Todorov I.T. Pepperkok R. Philipova R.N. Kearsey S.E. Ansorge W. Werner D. J. Cell Sci. 1994; 107: 253-265Crossref PubMed Google Scholar). Interaction between the MCM proteins has been reported in yeast (4Hennessy K.M. Lee A. Chen E. Botstein D. Genes Dev. 1991; 5: 958-969Crossref PubMed Scopus (229) Google Scholar, 7Yan H. Gibson S. Tye B.K. Genes Dev. 1991; 5: 944-957Crossref PubMed Scopus (152) Google Scholar), and the protein complexes containing MCM proteins are detected in Drosophila (8Su T.T. Feger G. O'Farrell P.H. Mol. Biol. Cell. 1996; 7: 319-326Crossref PubMed Scopus (48) Google Scholar), Xenopus(9Hendrickson M. Madine M. Dalton S. Gautier J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12223-12228Crossref PubMed Scopus (104) Google Scholar), mouse (10Kimura H. Takizawa N. Nozaki N. Sugimoto K. Nucleic Acids Res. 1995; 23: 2097-2104Crossref PubMed Scopus (67) Google Scholar, 11Kimura H. Ohtomo T. Yamaguchi M. Ishi A. Sugimoto K. Genes to Cells. 1996; 1: PubMed Scopus Google Scholar), and human cells D. R. R. J. Biochem. 1995; PubMed Scopus Google Scholar, R. D. R. J. Biochem. 1995; PubMed Scopus Google Scholar, M. T. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). human MCM4, -6, and -7 proteins a and MCM2 is associated with this complex D. R. R. J. Biochem. 1995; PubMed Scopus Google Scholar, D. A. R. R. J. Biochem. 1996; PubMed Scopus Google Scholar, H. R. 1996; PubMed Scopus Google Scholar). and proteins a complex (10Kimura H. Takizawa N. Nozaki N. Sugimoto K. Nucleic Acids Res. 1995; 23: 2097-2104Crossref PubMed Scopus (67) Google Scholar, R. D. R. J. Biochem. 1995; PubMed Scopus Google Scholar). All six of the MCM proteins DNA-dependent ATPase motifs in a central domain that is conserved from yeast to and these motifs are in that the DNA Nucleic Acids Res. 21: PubMed Scopus Google Scholar). These suggest that MCM protein complexes may be involved in the initiation of DNA replication as an that DNA in the origin DNA helicase activity of MCM proteins has been proteins with the initiation of DNA replication and are from the as DNA replication I.T. A. Kearsey S.E. J. Cell Biol. 1995; PubMed Scopus Google Scholar, H. Tye B.K. Genes Dev. 7: PubMed Scopus Google Scholar, Biol. 1995; 5: Full Text Full Text PDF PubMed Scopus Google Scholar). Our group that MCM protein complexes containing MCM2, -6, and -7 proteins with and were purified to by histone-Sepharose column chromatography S. A. Sato K. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the present it is that both ATPase and DNA helicase activities are associated with an MCM4, -6, and -7 protein that both ATPase and DNA helicase activities were present in the purified MCM protein The results of the immunodepletion using anti-MCM4 antibody suggested that the DNA helicase activity required the MCM protein complexes The of DNA helicase activity was detected at a of in the equal amounts of MCM4, -6, and -7 proteins were that a complex of MCM4, -6, and -7 is has the DNA helicase activity and it was suggested that or protein was of ATP The protein of the that these MCM proteins a of and the between the of this 600-kDa complex and the DNA helicase activity suggests that this complex as a DNA is that MCM4, -6, and -7 proteins a complex and that this complex with to a complex. The complex may be complexes of MCM proteins in the and was detected at a MCM proteins were the a complex was detected These results suggest that of MCM proteins are present in purified MCM proteins and that to of as a DNA DNA helicase activity was detected only in the MCM2 it is that the presence of MCM2 protein the of MCM4, -6, and -7 proteins to the DNA helicase MCM proteins of MCM2, -6, and -7 were to a histone-Sepharose column S. A. Sato K. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Our group that these only MCM2 protein has an for and H. Kimura, that a complex consisting of MCM2, -6, and -7 was to the histone-Sepharose column the between MCM2 and and a complex of MCM4, -6, and -7 was from MCM2 protein the of the MCM proteins with group D. R. R. J. Biochem. 1995; PubMed Scopus Google Scholar, D. A. R. R. J. Biochem. 1996; PubMed Scopus Google Scholar, H. R. 1996; PubMed Scopus Google has reported that MCM4, -6, and -7 proteins a complex and that MCM2 protein is associated with the of MCM proteins. that a of MCM4, -6, and -7 has DNA helicase activity is with the that J. T. J. J. Biol. Chem. Full Text PDF PubMed Google as a Biochem. 1996; PubMed Scopus Google Scholar, Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar). a that to MCM proteins has the DNA helicase activity be DNA helicase activity of MCM proteins be from the that of these proteins have motifs that are present in the proteins DNA-dependent ATPase activity Nucleic Acids Res. 21: PubMed Scopus Google Scholar). The DNA helicase activity may be for the initiation as as the of DNA MCM proteins are in the replication are with replication proteins of and replication protein single-stranded or DNA I.T. A. Kearsey S.E. J. Cell Biol. 1995; PubMed Scopus Google Scholar, Biol. 1995; 5: Full Text Full Text PDF PubMed Scopus Google H. Nozaki N. Sugimoto K. EMBO J. 1994; PubMed Scopus Google Scholar, T. R. J. Cell Sci. 1996; PubMed Google Scholar). MCM proteins in at the and from it as the DNA replication I.T. A. Kearsey S.E. J. Cell Biol. 1995; PubMed Scopus Google Scholar, Biol. 1995; 5: Full Text Full Text PDF PubMed Scopus Google Scholar, H. Nozaki N. Sugimoto K. EMBO J. 1994; PubMed Scopus Google Scholar, M. E. K. J. Cell Sci. 1995; Google Scholar). These with suggest that MCM proteins are required at an of the DNA replication as an that DNA in the origin of DNA have been in by the activity that displaces annealed oligonucleotides P. U. PubMed Scopus (48) Google Scholar, BioEssay. 1994; PubMed Scopus Google Scholar). a mouse DNA helicase M. T. T. S. J. T. M. T. Mol. Cell. Biol. 1995; 15: PubMed Scopus Google and Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google are for the involved in the of the replication The complex of MCM4, -6, and -7 proteins is from DNA in that it oligonucleotides the activity of DNA helicase in the fraction was with DNA of the MCM4, -6, and -7 protein complex may the DNA helicase or replication proteins as replication protein may be involved in the of DNA unwinding by the MCM protein complex. is that the DNA helicase activity of MCM proteins is required only at the of DNA unwinding of the origin and DNA the role of MCM proteins in the DNA origin recognition complex consisting of six was in yeast as a complex that the origin PubMed Scopus Google Scholar), and it has been that the is required for DNA replication R. PubMed Scopus Google Scholar, M. P. J. PubMed Scopus Google Scholar, G. A. J. K. J. PubMed Scopus Google Scholar, S. J. R. S. Mol. Biol. Cell. 1995; PubMed Scopus Google Scholar). of A. J.P.J. M. Blow J.J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google and 1996; PubMed Scopus Google were in and it was that are required for DNA replication in an in between and MCM proteins has been reported in S. S. J. R. S. Mol. Biol. Cell. 1995; PubMed Scopus Google Scholar, J.J. PubMed Scopus Google Scholar). MCM protein complexes may be involved in the initiation of DNA replication as a DNA unwinding by with is that MCM proteins may MCM protein complexes of MCM2, -6, and -7 have a for in which was S. A. Sato K. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The that hydrolyzable ATP is required for the DNA helicase activity of MCM proteins with the that ATP is required for DNA replication in the J.P.J. Blow J.J. 1995; PubMed Scopus Google Scholar). this both licensing and are required for DNA has been purified to and of a complex of MCM proteins J.P.J. Blow J.J. 1995; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar, S. S. H. H. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). has been suggested that is required for MCM proteins to J.P.J. Blow J.J. 1995; PubMed Scopus Google Scholar). Cell. 1996; Full Text Full Text PDF PubMed Scopus Google have reported that the of and proteins to is required for the of MCM protein with which to the initiation of DNA replication in the the present it was that both ATPase and DNA helicase activities were associated with purified MCM proteins. the of these activities are of MCM4, -6, and -7 protein complex this the evidence for the that MCM protein complexes as a DNA unwinding in the initiation of DNA There are at least six MCM 1The abbreviations used are: MCM, minichromosome maintenance; DTT, dithiothreitol; DSP, dithiobis-succinimidyl propionate; ATPγS, adenosine 5′-O-(thiotriphosphate); ORC, origin recognition complex; AMP-PNP, β-γ-imidoadenosine-5′-triphosphate; RLF, replication licensing factor.1The abbreviations used are: MCM, minichromosome maintenance; DTT, dithiothreitol; DSP, dithiobis-succinimidyl propionate; ATPγS, adenosine 5′-O-(thiotriphosphate); ORC, origin recognition complex; AMP-PNP, β-γ-imidoadenosine-5′-triphosphate; RLF, replication licensing factor. proteins that play an essential role in eukaryotic DNA replication as follows: MCM2, -3, -4 (Cdc21), -5, -6 (Mis5), and -7 (CDC47) proteins (see reviews in Refs.1Tye B.-K. Trends Cell Biol. 1994; 4: 160-166Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 2Chong J.P.J. Thömmes P. Blow J.J. Trends Biochem. Sci. 1996; 21: 102-106Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 3Kearsey S.E. Mariorano D. Holmes E.C. Todorov I.T. BioEssay. 1996; 18: 183-190Crossref PubMed Scopus (120) Google Scholar). Genetic evidence suggests that these proteins are required for the initiation of DNA replication in yeast (4Hennessy K.M. Lee A. Chen E. Botstein D. Genes Dev. 1991; 5: 958-969Crossref PubMed Scopus (229) Google Scholar, 5Mariorano D. Blom van Assendelft G. Kearsey S.E. EMBO J. 1996; 15: 861-872Crossref PubMed Scopus (83) Google Scholar). The inactivation of human MCM2 protein with antibody consistently inhibits the initiation of DNA replication (6Todorov I.T. Pepperkok R. Philipova R.N. Kearsey S.E. Ansorge W. Werner D. J. Cell Sci. 1994; 107: 253-265Crossref PubMed Google Scholar). Interaction between the MCM proteins has been reported in yeast (4Hennessy K.M. Lee A. Chen E. Botstein D. Genes Dev. 1991; 5: 958-969Crossref PubMed Scopus (229) Google Scholar, 7Yan H. Gibson S. Tye B.K. Genes Dev. 1991; 5: 944-957Crossref PubMed Scopus (152) Google Scholar), and the protein complexes containing MCM proteins are detected in Drosophila (8Su T.T. Feger G. O'Farrell P.H. Mol. Biol. Cell. 1996; 7: 319-326Crossref PubMed Scopus (48) Google Scholar), Xenopus(9Hendrickson M. Madine M. Dalton S. Gautier J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12223-12228Crossref PubMed Scopus (104) Google Scholar), mouse (10Kimura H. Takizawa N. Nozaki N. Sugimoto K. Nucleic Acids Res. 1995; 23: 2097-2104Crossref PubMed Scopus (67) Google Scholar, 11Kimura H. Ohtomo T. Yamaguchi M. Ishi A. Sugimoto K. Genes to Cells. 1996; 1: PubMed Scopus Google Scholar), and human cells D. R. R. J. Biochem. 1995; PubMed Scopus Google Scholar, R. D. R. J. Biochem. 1995; PubMed Scopus Google Scholar, M. T. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). human MCM4, -6, and -7 proteins a and MCM2 is associated with this complex D. R. R. J. Biochem. 1995; PubMed Scopus Google Scholar, D. A. R. R. J. Biochem. 1996; PubMed Scopus Google Scholar, H. R. 1996; PubMed Scopus Google Scholar). and proteins a complex (10Kimura H. Takizawa N. Nozaki N. Sugimoto K. Nucleic Acids Res. 1995; 23: 2097-2104Crossref PubMed Scopus (67) Google Scholar, R. D. R. J. Biochem. 1995; PubMed Scopus Google Scholar). All six of the MCM proteins DNA-dependent ATPase motifs in a central domain that is conserved from yeast to and these motifs are in that the DNA Nucleic Acids Res. 21: PubMed Scopus Google Scholar). These suggest that MCM protein complexes may be involved in the initiation of DNA replication as an that DNA in the origin DNA helicase activity of MCM proteins has been MCM proteins with the initiation of DNA replication and are from the as DNA replication I.T. A. Kearsey S.E. J. Cell Biol. 1995; PubMed Scopus Google Scholar, H. Tye B.K. Genes Dev. 7: PubMed Scopus Google Scholar, Biol. 1995; 5: Full Text Full Text PDF PubMed Scopus Google Scholar). Our group that MCM protein complexes containing MCM2, -6, and -7 proteins with and were purified to by histone-Sepharose column chromatography S. A. Sato K. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the present it is that both ATPase and DNA helicase activities are associated with an MCM4, -6, and -7 protein complex. that both ATPase and DNA helicase activities were present in the purified MCM protein The results of the immunodepletion using anti-MCM4 antibody suggested that the DNA helicase activity required the MCM protein complexes The of DNA helicase activity was detected at a of in the equal amounts of MCM4, -6, and -7 proteins were that a complex of MCM4, -6, and -7 is has the DNA helicase activity and it was suggested that or protein was of ATP The protein of the that these MCM proteins a of and the between the of this 600-kDa complex and the DNA helicase activity suggests that this complex as a DNA is that MCM4, -6, and -7 proteins a complex and that this complex with to a complex. The complex may be complexes of MCM proteins in the and was detected at a MCM proteins were the a complex was detected These results suggest that of MCM proteins are present in purified MCM proteins and that to of as a DNA DNA helicase activity was detected only in the MCM2 it is that the presence of MCM2 protein the of MCM4, -6, and -7 proteins to the DNA helicase MCM proteins of MCM2, -6, and -7 were to a histone-Sepharose column S. A. Sato K. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Our group that these only MCM2 protein has an for and H. Kimura, that a complex consisting of MCM2, -6, and -7 was to the histone-Sepharose column the between MCM2 and and a complex of MCM4, -6, and -7 was from MCM2 protein the of the MCM proteins with group D. R. R. J. Biochem. 1995; PubMed Scopus Google Scholar, D. A. R. R. J. Biochem. 1996; PubMed Scopus Google Scholar, H. R. 1996; PubMed Scopus Google has reported that MCM4, -6, and -7 proteins a complex and that MCM2 protein is associated with the of MCM proteins. that a of MCM4, -6, and -7 has DNA helicase activity is with the that J. T. J. J. Biol. Chem. Full Text PDF PubMed Google as a Biochem. 1996; PubMed Scopus Google Scholar, Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar). a that to MCM proteins has the DNA helicase activity be DNA helicase activity of MCM proteins be from the that of these proteins have motifs that are present in the proteins DNA-dependent ATPase activity Nucleic Acids Res. 21: PubMed Scopus Google Scholar). The DNA helicase activity may be for the initiation as as the of DNA MCM proteins are in the replication are with replication proteins of and replication protein single-stranded or DNA I.T. A. Kearsey S.E. J. Cell Biol. 1995; PubMed Scopus Google Scholar, Biol. 1995; 5: Full Text Full Text PDF PubMed Scopus Google H. Nozaki N. Sugimoto K. EMBO J. 1994; PubMed Scopus Google Scholar, T. R. J. Cell Sci. 1996; PubMed Google Scholar). MCM proteins in at the and from it as the DNA replication I.T. A. Kearsey S.E. J. Cell Biol. 1995; PubMed Scopus Google Scholar, Biol. 1995; 5: Full Text Full Text PDF PubMed Scopus Google Scholar, H. Nozaki N. Sugimoto K. EMBO J. 1994; PubMed Scopus Google Scholar, M. E. K. J. Cell Sci. 1995; Google Scholar). These with suggest that MCM proteins are required at an of the DNA replication as an that DNA in the origin of DNA have been in by the activity that displaces annealed oligonucleotides P. U. PubMed Scopus (48) Google Scholar, BioEssay. 1994; PubMed Scopus Google Scholar). a mouse DNA helicase M. T. T. S. J. T. M. T. Mol. Cell. Biol. 1995; 15: PubMed Scopus Google and Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google are for the involved in the of the replication The complex of MCM4, -6, and -7 proteins is from DNA in that it oligonucleotides the activity of DNA helicase in the fraction was with DNA of the MCM4, -6, and -7 protein complex may the DNA helicase or replication proteins as replication protein may be involved in the of DNA unwinding by the MCM protein complex. is that the DNA helicase activity of MCM proteins is required only at the of DNA unwinding of the origin and DNA the role of MCM proteins in the DNA origin recognition complex consisting of six was in yeast as a complex that the origin PubMed Scopus Google Scholar), and it has been that the is required for DNA replication R. PubMed Scopus Google Scholar, M. P. J. PubMed Scopus Google Scholar, G. A. J. K. J. PubMed Scopus Google Scholar, S. J. R. S. Mol. Biol. Cell. 1995; PubMed Scopus Google Scholar). of A. J.P.J. M. Blow J.J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google and 1996; PubMed Scopus Google were in and it was that are required for DNA replication in an in between and MCM proteins has been reported in S. S. J. R. S. Mol. Biol. Cell. 1995; PubMed Scopus Google Scholar, J.J. PubMed Scopus Google Scholar). MCM protein complexes may be involved in the initiation of DNA replication as a DNA unwinding by with is that MCM proteins may MCM protein complexes of MCM2, -6, and -7 have a for in which was S. A. Sato K. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The that hydrolyzable ATP is required for the DNA helicase activity of MCM proteins with the that ATP is required for DNA replication in the J.P.J. Blow J.J. 1995; PubMed Scopus Google Scholar). this both licensing and are required for DNA has been purified to and of a complex of MCM proteins J.P.J. Blow J.J. 1995; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar, S. S. H. H. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). has been suggested that is required for MCM proteins to J.P.J. Blow J.J. 1995; PubMed Scopus Google Scholar). Cell. 1996; Full Text Full Text PDF PubMed Scopus Google have reported that the of and proteins to is required for the of MCM protein with which to the initiation of DNA replication in the the present it was that both ATPase and DNA helicase activities were associated with purified MCM proteins. the of these activities are of MCM4, -6, and -7 protein complex this the evidence for the that MCM protein complexes as a DNA unwinding in the initiation of DNA that both ATPase and DNA helicase activities were present in the purified MCM protein The results of the immunodepletion using anti-MCM4 antibody suggested that the DNA helicase activity required the MCM protein complexes The of DNA helicase activity was detected at a of in the equal amounts of MCM4, -6, and -7 proteins were that a complex of MCM4, -6, and -7 is has the DNA helicase activity and it was suggested that or protein was of ATP The protein of the that these MCM proteins a of and the between the of this 600-kDa complex and the DNA helicase activity suggests that this complex as a DNA is that MCM4, -6, and -7 proteins a complex and that this complex with to a complex. The complex may be complexes of MCM proteins in the and was detected at a MCM proteins were the a complex was detected These results suggest that of MCM proteins are present in purified MCM proteins and that to of as a DNA DNA helicase activity was detected only in the MCM2 it is that the presence of MCM2 protein the of MCM4, -6, and -7 proteins to the DNA helicase activity. The MCM proteins of MCM2, -6, and -7 were to a histone-Sepharose column S. A. Sato K. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Our group that these only MCM2 protein has an for and H. Kimura, that a complex consisting of MCM2, -6, and -7 was to the histone-Sepharose column the between MCM2 and and a complex of MCM4, -6, and -7 was from MCM2 protein the of the MCM proteins with group D. R. R. J. Biochem. 1995; PubMed Scopus Google Scholar, D. A. R. R. J. Biochem. 1996; PubMed Scopus Google Scholar, H. R. 1996; PubMed Scopus Google has reported that MCM4, -6, and -7 proteins a complex and that MCM2 protein is associated with the of MCM proteins. that a of MCM4, -6, and -7 has DNA helicase activity is with the that J. T. J. J. Biol. Chem. Full Text PDF PubMed Google as a Biochem. 1996; PubMed Scopus Google Scholar, Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar). a that to MCM proteins has the DNA helicase activity be The DNA helicase activity of MCM proteins be from the that of these proteins have motifs that are present in the proteins DNA-dependent ATPase activity Nucleic Acids Res. 21: PubMed Scopus Google Scholar). The DNA helicase activity may be for the initiation as as the of DNA MCM proteins are in the replication are with replication proteins of and replication protein single-stranded or DNA I.T. A. Kearsey S.E. J. Cell Biol. 1995; PubMed Scopus Google Scholar, Biol. 1995; 5: Full Text Full Text PDF PubMed Scopus Google H. Nozaki N. Sugimoto K. EMBO J. 1994; PubMed Scopus Google Scholar, T. R. J. Cell Sci. 1996; PubMed Google Scholar). MCM proteins in at the and from it as the DNA replication I.T. A. Kearsey S.E. J. Cell Biol. 1995; PubMed Scopus Google Scholar, Biol. 1995; 5: Full Text Full Text PDF PubMed Scopus Google Scholar, H. Nozaki N. Sugimoto K. EMBO J. 1994; PubMed Scopus Google Scholar, M. E. K. J. Cell Sci. 1995; Google Scholar). These with suggest that MCM proteins are required at an of the DNA replication as an that DNA in the origin of DNA have been in by the activity that displaces annealed oligonucleotides P. U. PubMed Scopus (48) Google Scholar, BioEssay. 1994; PubMed Scopus Google Scholar). a mouse DNA helicase M. T. T. S. J. T. M. T. Mol. Cell. Biol. 1995; 15: PubMed Scopus Google and Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google are for the involved in the of the replication The complex of MCM4, -6, and -7 proteins is from DNA in that it oligonucleotides the activity of DNA helicase in the fraction was with DNA of the MCM4, -6, and -7 protein complex may the DNA helicase or replication proteins as replication protein may be involved in the of DNA unwinding by the MCM protein complex. is that the DNA helicase activity of MCM proteins is required only at the of DNA unwinding of the origin and DNA the role of MCM proteins in the DNA The origin recognition complex consisting of six was in yeast as a complex that the origin PubMed Scopus Google Scholar), and it has been that the is required for DNA replication R. PubMed Scopus Google Scholar, M. P. J. PubMed Scopus Google Scholar, G. A. J. K. J. PubMed Scopus Google Scholar, S. J. R. S. Mol. Biol. Cell. 1995; PubMed Scopus Google Scholar). of A. J.P.J. M. Blow J.J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google and 1996; PubMed Scopus Google were in and it was that are required for DNA replication in an in between and MCM proteins has been reported in S. S. J. R. S. Mol. Biol. Cell. 1995; PubMed Scopus Google Scholar, J.J. PubMed Scopus Google Scholar). MCM protein complexes may be involved in the initiation of DNA replication as a DNA unwinding by with is that MCM proteins may MCM protein complexes of MCM2, -6, and -7 have a for in which was S. A. Sato K. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The that hydrolyzable ATP is required for the DNA helicase activity of MCM proteins with the that ATP is required for DNA replication in the J.P.J. Blow J.J. 1995; PubMed Scopus Google Scholar). this both licensing and are required for DNA has been purified to and of a complex of MCM proteins J.P.J. Blow J.J. 1995; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar, S. S. H. H. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). has been suggested that is required for MCM proteins to J.P.J. Blow J.J. 1995; PubMed Scopus Google Scholar). Cell. 1996; Full Text Full Text PDF PubMed Scopus Google have reported that the of and proteins to is required for the of MCM protein with which to the initiation of DNA replication in the the present it was that both ATPase and DNA helicase activities were associated with purified MCM proteins. the of these activities are of MCM4, -6, and -7 protein complex this the evidence for the that MCM protein complexes as a DNA unwinding in the initiation of DNA for the anti-MCM4 antibody and for for mouse DNA helicase for in ATPase and for for the DNA helicase activity is associated with an MCM4, -6, and -7 protein of The of The in a of DTT, of and of MCM proteins purified by or DNA PDF