复制蛋白A
同源重组
非同源性末端接合
核酸酶
解旋酶
重组DNA
DNA修复
生物
DNA
DNA修复蛋白XRCC4
细胞生物学
雷达50
分子生物学
遗传学
核苷酸切除修复
DNA结合蛋白
基因
核糖核酸
转录因子
作者
Roopesh Anand,Cosimo Pinto,Petr Ćejka
出处
期刊:Methods in Enzymology
日期:2018-01-01
卷期号:: 25-66
被引量:28
标识
DOI:10.1016/bs.mie.2017.11.008
摘要
Accurate repair of DNA double-strand breaks (DSBs) is carried out by homologous recombination. In order to repair DNA breaks by the recombination pathway, the 5'-terminated DNA strand at DSB sites must be first nucleolytically processed to produce 3'-overhang. The process is termed DNA end resection and involves the interplay of several nuclease complexes. DNA end resection commits DSB repair to the recombination pathway including a process termed single-strand annealing, as resected DNA ends are generally nonligatable by the competing nonhomologous end-joining machinery. Biochemical reconstitution experiments provided invaluable mechanistic insights into the DNA end resection pathways. In this chapter, we describe preparation procedures of key proteins involved in DNA end resection in human cells, including the MRE11-RAD50-NBS1 complex, phosphorylated variant of CtIP, the DNA2 nuclease-helicase with its helicase partners Bloom (BLM) or Werner (WRN), as well as the single-stranded DNA-binding protein replication protein A. The availability of recombinant DNA end resection factors will help to further elucidate resection mechanisms and regulatory processes that may involve novel protein partners and posttranslational modifications.
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