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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication
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A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication

机译：真核DNA复制许可期间，将双六聚体MCM2-7复合物加载到原始DNA上

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摘要

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 Saccha-romyces 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.
机译：在复制前复合体（pre-RC）形成过程中，起源识别复合体（ORC），Cdc6和Cdt1将6个亚单位的微型染色体维持复合体（MCM2-7）协同加载到DNA上。加载MCM2-7是DNA许可的先决条件，该许可将DNA复制限制为每个细胞周期一次。在S阶段，MCM2-7充当复制解旋酶的一部分，但在RC前MCM2-7处于非激活状态。已经在细菌和病毒中研究了复制性DNA解旋酶的前后结构，而在真核生物中没有研究过复制性DNA解旋酶的结构，这对于理解MCM2-7的加载机制和复制体组装具有重要意义。长期以来，缺乏有效的重构前RC系统阻碍了MCM2-7加载的详细机理和结构分析。我们已经用纯化的蛋白质重建了酿酒酵母前RC的形成，并显示了MCM2-7在体外有效装载到原始DNA上。发现MCM2-7的上载取决于所有pre-RC蛋白，起源DNA和ATP水解的存在。 MCM2-7的四级结构在RC前形成期间会发生变化：加载前的MCM2-7是溶液中的单一六聚体，但在RC前形成期间会转变为双六聚体。使用电子显微镜（EM），我们观察到加载的MCM2-7环绕着DNA。加载的MCM2-7复合物可以在DNA上滑动，并且滑动不是定向的。我们的结果提供了对前RC形成机制的关键见解，并对理解MCM2-7在建立双向复制叉中的作用具有重要意义。

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    Cecile Evrin; Pippa Clarke; Juergen Zech; Rudi Lurz; Jingchuan Sun; Stefan Uhle; Huilin Li; Bruce Stillman; Christian Speck;

  • 作者单位

    DNA Replication Group, Medical Research Council Clinical Sciences Centre, Imperial College London, London W12 ONN, United Kingdom;

    DNA Replication Group, Medical Research Council Clinical Sciences Centre, Imperial College London, London W12 ONN, United Kingdom;

    DNA Replication Group, Medical Research Council Clinical Sciences Centre, Imperial College London, London W12 ONN, United Kingdom;

    Microscopy Unit, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany;

    Biology Department, Brookhaven National Laboratory, Upton, NY 11973;

    DNA Replication Group, Medical Research Council Clinical Sciences Centre, Imperial College London, London W12 ONN, United Kingdom MBM ScienceBridge GmbH, Hans-Adolf-Krebs-Weg 1, 37077 Gottingen, Germany;

    Biology Department, Brookhaven National Laboratory, Upton, NY 11973;

    Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724;

    DNA Replication Group, Medical Research Council Clinical Sciences Centre, Imperial College London, London W12 ONN, United Kingdom;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    helicase; initiation; mini chromosome maintenance; ORC; pre-RC;

    机译：解旋酶引发;小染色体维持;ORC;前RC;

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