用于检测同源重组中修复DNA合成的重组系统。
Reconstituted System for the Examination of Repair DNA Synthesis in Homologous Recombination.
作者信息
Kwon Youngho, Daley James M, Sung Patrick
机构信息
Yale University School of Medicine, New Haven, CT, United States.
Yale University School of Medicine, New Haven, CT, United States.
出版信息
Methods Enzymol. 2017;591:307-325. doi: 10.1016/bs.mie.2017.03.021. Epub 2017 Apr 21.
Abstract
In homologous recombination (HR), DNA polymerase δ-mediated DNA synthesis occurs within the displacement loop (D-loop) that is made by the recombinase Rad51 in conjunction with accessory factors. We describe in this chapter the reconstitution of the D-loop and repair DNA synthesis reactions using purified Saccharomyces cerevisiae HR (Rad51, RPA, and Rad54) and DNA replication (PCNA, RFC, and DNA polymerase δ) proteins and document the role of the Pif1 helicase in DNA synthesis via a migrating DNA bubble intermediate. These reconstituted systems are particularly valuable for understanding the conserved mechanism of repair DNA synthesis dependent on DNA polymerase δ and its cognate helicase in eukaryotic organisms.
摘要
在同源重组（HR）过程中，DNA聚合酶δ介导的DNA合成发生在由重组酶Rad51与辅助因子共同形成的置换环（D环）内。在本章中，我们描述了使用纯化的酿酒酵母HR（Rad51、RPA和Rad54）和DNA复制（PCNA、RFC和DNA聚合酶δ）蛋白重建D环和修复DNA合成反应，并通过迁移的DNA泡中间体记录了Pif1解旋酶在DNA合成中的作用。这些重建系统对于理解真核生物中依赖DNA聚合酶δ及其同源解旋酶的修复DNA合成的保守机制特别有价值。
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