真核生物前导链/后随链DNA复制的检测方法。
Assays for Eukaryotic Leading/Lagging Strand DNA Replication.
作者信息
Schauer Grant, Finkelstein Jeff, O'Donnell Mike
机构信息
Howard Hughes Medical Institute, Rockefeller University, New York, USA.
出版信息
Bio Protoc. 2017 Sep 20;7(18). doi: 10.21769/BioProtoc.2548.
Abstract
The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their 'correct' strands, as well as quality control mechanisms that evict polymerases when they associate with an 'incorrect' strand. Here, we provide a practical guide to differentially assay leading and lagging strand replication using pure proteins.
摘要
真核生物复制体是一种复制DNA的多蛋白复合体。复制体经过精心构建，以将连续的前导链合成与不连续的后随链合成偶联起来，这主要分别由DNA聚合酶ε和δ，以及解旋酶、聚合酶α-引发酶、DNA滑动夹、夹装载器和许多其他蛋白质来完成。我们之前已经确立了聚合酶ε和δ靶向其“正确”链的机制，以及当聚合酶与“错误”链结合时将其逐出的质量控制机制。在此，我们提供一份实用指南，介绍如何使用纯蛋白对前导链和后随链复制进行差异检测。
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本文引用的文献
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2
Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale.在毫秒时间尺度上解析冈崎片段成熟的各个步骤。
Nat Struct Mol Biol. 2016 May;23(5):402-8. doi: 10.1038/nsmb.3207. Epub 2016 Apr 11.
3
Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation.真核生物复制体的重组揭示了定义前导链/后随链运作的抑制机制。
Elife. 2015 Apr 14;4:e04988. doi: 10.7554/eLife.04988.
4
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5
Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork.真核复制叉处不对称聚合酶组装的机制。
Nat Struct Mol Biol. 2014 Aug;21(8):664-70. doi: 10.1038/nsmb.2851. Epub 2014 Jul 6.
6
A solution to release twisted DNA during chromosome replication by coupled DNA polymerases.通过偶联 DNA 聚合酶在染色体复制过程中释放扭曲的 DNA 的解决方案。
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7
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