A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication
A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication
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DOI：
10.1073/pnas.0911500106
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发表时间：
2009-12-01
影响因子：
11.1
通讯作者：
Speck, Christian
中科院分区：
文献类型：
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作者：
Evrin, Cecile;Clarke, Pippa;Speck, Christian
在复制前复合物（pre-RC）形成期间，起始识别复合物（ORC）、Cdc 6和Cdt 1协同将6亚基微型染色体维持（MCM 2 -7）复合物加载到DNA上。MCM 2 -7的加载是DNA许可的先决条件，DNA许可将DNA复制限制为每个细胞周期一次。在S期，MCM 2 -7作为复制解旋酶的一部分发挥作用，但在RC前，MCM 2 -7无活性。复制DNA解旋酶在装载到DNA上之前和之后的组织已经在细菌和病毒中进行了研究，但不是真核生物，并且对于理解MCM 2 -7装载机制和复制体组装非常重要。长期以来，由于缺乏有效的预制钢筋混凝土系统，对MCM 2 -7加载的详细机理和结构分析受到阻碍。我们已经用纯化的蛋白质重建了酿酒酵母前RC形成，并在体外显示出将MCM 2 -7有效地加载到原始DNA上。发现MCM 2 -7负载依赖于所有前RC蛋白、来源DNA和ATP水解的存在。在预RC形成期间，MCM 2 -7的四级结构发生变化：在加载之前，MCM 2 -7在溶液中是单个六聚体，但在预RC形成期间转化为双六聚体。使用电子显微镜（EM），我们观察到负载的MCM 2 -7包围DNA。加载的MCM 2 -7复合物可以在DNA上滑动，并且滑动不是定向的。我们的研究结果提供了关键的见解前RC形成的机制，并有重要的意义，了解MCM 2 -7在建立双向复制叉的作用。
During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.