Biochemical activities associated with mouse Mcm2 protein.
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.
Ishimi
Y
Y
Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan. yukio@libra.ls.m-kagaku.co.jp
Komamura-Kohno
Y
Y
Arai
K
K
Masai
H
H
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D064111:Minichromosome Maintenance Complex Component 2; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism* / Q000502:physiology*; D009707:Nucleosomes / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D009707:Nucleosomes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; C068619:CDC7 protein, human; C489403:Cdc7 protein, mouse; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; D064111:Minichromosome Maintenance Complex Component 2