Molecular analysis and regulation of replication fork complex, especially focus on MCM helicase

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Mini-chromosome maintenance(Mcm) is a central component for DNA unwinding reaction during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and polymeraseα-primase complexes, we find that the Mcm4/6/7 complex co-sediments with the polymeraseα-primase complex in the glycerol gradient centrifugation. A direct physical interaction between the Mcm2～ 7 complex and human DNA polymeraseα-primase subunits p48-p58 hetero-dimer is detected in pull-down assay. Single-stranded DNA binding activities of both primase and Mcm2～ 7 increase when they coexist. Both the Mcm4/6/7 and Mcm2～ 7 complexes stimulate primer RNA synthesis activity of primase in vitro. However, the helicase and ATP hydrolysis activities of Mcm4/6/7 are not affected by primase. These results indicate a possibility that a direct physical interaction between primase and Mcm may activate priming reaction by the former protein.