真核生物DNA复制过程中MCM2-7、Cdc45和GINS在DNA解旋位点的定位。

Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication.

作者信息

Pacek Marcin, Tutter Antonin V, Kubota Yumiko, Takisawa Haruhiko, Walter Johannes C

机构信息

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.

出版信息

Mol Cell. 2006 Feb 17;21(4):581-7. doi: 10.1016/j.molcel.2006.01.030.

DOI:10.1016/j.molcel.2006.01.030

PMID:16483939

Abstract

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.

摘要

关于真核生物DNA复制叉的结构和生化组成，我们所知甚少。为了研究这个问题，我们使用生物素-链霉亲和素修饰的质粒在非洲爪蟾卵提取物中诱导序列特异性复制叉暂停。采用染色质免疫沉淀法来鉴定与暂停叉相关的因子。这种方法确定了DNA聚合酶α、DNA聚合酶δ、DNA聚合酶ε、MCM2 - 7、Cdc45、GINS和Mcm10是脊椎动物复制体的组成成分。在DNA聚合酶抑制剂阿非科林存在的情况下，它会导致高度持续的DNA解旋酶与停滞的复制体解偶联，此时只有Cdc45、GINS和MCM2 - 7在暂停位点富集。数据表明存在一种大分子机器，即“解旋体”，它在复制叉处分离DNA链，并包含Cdc45、GINS和MCM2 - 7全复合体。