Fien Karen, Cho Young-Sik, Lee Joon-Kyu, Raychaudhuri Santanu, Tappin Inger, Hurwitz Jerard
Program in Molecular Biology, Memorial-Sloan Kettering Cancer Center, New York, New York 10021, USA.
J Biol Chem. 2004 Apr 16;279(16):16144-53. doi: 10.1074/jbc.M400142200. Epub 2004 Feb 6.
Models of DNA replication in yeast and Xenopus suggest that Mcm10p is required to generate the pre-initiation complex as well as progression of the replication fork during the elongation of DNA chains. In this report, we show that the Schizosaccharomyces pombe Mcm10p/Cdc23p binds to the S. pombe DNA polymerase (pol) alpha-primase complex in vitro by interacting specifically with the catalytic p180 subunit and stimulates DNA synthesis catalyzed by the pol alpha-primase complex with various primed DNA templates. We investigated the mechanism by which Mcm10p activates the polymerase activity of the pol alpha-primase complex by generating truncated derivatives of the full-length 593-amino acid Mcm10p. Their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA and to pol alpha were compared. Concomitant with increased deletion of the N-terminal region (from amino acids 95 to 415), Mcm10p derivatives lost their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA. Truncated derivatives of Mcm10p containing amino acids 1-416 retained the pol alpha binding activity, whereas the C terminus, amino acids 496-593, did not. These results demonstrate that both the single-stranded DNA binding and the pol alpha binding properties of Mcm10p play important roles in the activation. In accord with these findings, Mcm10p facilitated the binding of pol alpha-primase complex to primed DNA and formed a stable complex with pol alpha-primase on primed templates. A mutant that failed to activate or bind to DNA and pol alpha, was not observed in this complex. We suggest that the interaction of Mcm10p with the pol alpha-primase complex, its binding to single-stranded DNA, and its activation of the polymerase complex together contribute to its role in the elongation phase of DNA replication.
酵母和非洲爪蟾的DNA复制模型表明，在DNA链延伸过程中，生成预起始复合物以及复制叉的推进都需要Mcm10p。在本报告中，我们发现粟酒裂殖酵母的Mcm10p/Cdc23p在体外通过与催化性p180亚基特异性相互作用，与粟酒裂殖酵母DNA聚合酶（pol）α-引发酶复合物结合，并刺激pol α-引发酶复合物在各种带引物的DNA模板上催化DNA合成。我们通过生成全长593个氨基酸的Mcm10p的截短衍生物，研究了Mcm10p激活pol α-引发酶复合物聚合酶活性的机制。比较了它们刺激pol α聚合酶活性、结合单链DNA以及与pol α结合的能力。随着N端区域（从第95位氨基酸到第415位氨基酸）缺失的增加，Mcm10p衍生物失去了刺激pol α聚合酶活性和结合单链DNA的能力。包含第1 - 416位氨基酸的Mcm10p截短衍生物保留了与pol α的结合活性，而C端（第496 - 593位氨基酸）则没有。这些结果表明，Mcm10p的单链DNA结合特性和与pol α的结合特性在激活过程中都起着重要作用。与这些发现一致的是，Mcm10p促进了pol α-引发酶复合物与带引物DNA的结合，并在带引物的模板上与pol α-引发酶形成了稳定的复合物。在该复合物中未观察到未能激活或结合DNA及pol α的突变体。我们认为，Mcm10p与pol α-引发酶复合物的相互作用、其与单链DNA的结合以及对聚合酶复合物的激活共同促成了其在DNA复制延伸阶段的作用。
