Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication
Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication
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DOI：
10.1016/j.molcel.2006.01.030
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发表时间：
2006-02-17
期刊：
影响因子：
16
通讯作者：
Walter, JC
中科院分区：
文献类型：
--
作者：
Pacek, M;Tutter, AV;Walter, JC
对真核生物DNA复制叉的结构和生化组成知之甚少。为了研究这一问题，我们使用生物素-链亲和素修饰的质粒在爪蟾卵提取物中诱导序列特异性复制叉暂停。染色质免疫沉淀法用于鉴定与暂停叉相关的因素。该方法鉴定了DNA pol α、DNA pol δ、DNA pol epsilon、MCM2-7、Cdc45、GINS和Mcm10作为脊椎动物复制体的组成部分。在DNA聚合酶抑制剂aphidicolin存在的情况下，只有Cdc45、GINS和MCM2-7在暂停位点富集。aphidicolin会导致高度进展的DNA解旋酶从停滞的复制体上解偶联。这些数据表明存在一种大型分子机器，即“解窗体”，它在复制叉上分离DNA链，包含Cdc45、GINS和MCM2-7全息复合物。
Little is known about the architecture and biochemical composition of the eukaryotic DNA replication fork. To study this problem, we used biotin-streptavidin-modified plasmids to induce sequence-specific replication fork pausing in Xenopus egg extracts. Chromatin immunoprecipitation was employed to identify factors associated with the paused fork. This approach identifies DNA pol alpha, DNA pol delta, DNA pol epsilon, MCM2-7, Cdc45, GINS, and Mcm10 as components of the vertebrate replisome. In the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45, GINS, and MCM2-7 are enriched at the pause site. The data suggest the existence of a large molecular machine, the "unwindosome," which separates DNA strands at the replication fork and contains Cdc45, GINS, and the MCM2-7 holocomplex.