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

微染色体维持（Mcm）是真核生物DNA复制过程中DNA解绕反应的核心组成部分。DNA引物酶的活性需要在DNA复制叉合成短RNA引物，以延长DNA链上的滞后链。虽然在许多原核生物和病毒系统中，解旋酶和引物酶之间的直接物理和功能相互作用是已知的，但在真核生物中尚未探索解旋酶和引物酶之间的潜在相互作用。使用纯化的Mcm和聚合酶α-引物酶复合物，我们发现Mcm4/6/7复合物在甘油梯度离心中与聚合酶α-引物酶复合物共沉淀。Mcm2~ 7复合物与人DNA聚合酶α-引物酶亚基p48-p58异二聚体之间存在直接的物理相互作用。引物酶和Mcm2~ 7共存时单链DNA结合活性增加。Mcm2 /6/7和Mcm2~ 7复合物均能刺激引物酶的体外引物RNA合成活性。而Mcm4/6/7的解旋酶和ATP水解活性不受引物酶的影响。这些结果表明，引物酶和Mcm之间的直接物理相互作用可能会激活前者蛋白的引物反应。

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.