Schauer Grant, Finkelstein Jeff, O'Donnell Mike
Howard Hughes Medical Institute, Rockefeller University, New York, USA.
Bio Protoc. 2017 Sep 20;7(18). doi: 10.21769/BioProtoc.2548.
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.
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。
