Anand Roopesh, Pinto Cosimo, Cejka Petr
Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland.
Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland.
Methods Enzymol. 2018;600:25-66. doi: 10.1016/bs.mie.2017.11.008. Epub 2018 Feb 1.
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.
DNA双链断裂（DSB）的精确修复是通过同源重组来进行的。为了通过重组途径修复DNA断裂，DSB位点处5'端终止的DNA链必须首先经过核酸酶处理以产生3'突出端。这个过程被称为DNA末端切除，涉及几种核酸酶复合物的相互作用。DNA末端切除使DSB修复进入重组途径，包括一个称为单链退火的过程，因为切除的DNA末端通常不能被竞争性的非同源末端连接机制连接。生化重建实验为DNA末端切除途径提供了宝贵的机制见解。在本章中，我们描述了人类细胞中参与DNA末端切除的关键蛋白质的制备程序，包括MRE11-RAD50-NBS1复合物、CtIP的磷酸化变体、与解旋酶伙伴布鲁姆（BLM）或沃纳（WRN）结合的DNA2核酸酶-解旋酶，以及单链DNA结合蛋白复制蛋白A。重组DNA末端切除因子的可用性将有助于进一步阐明可能涉及新型蛋白质伙伴和翻译后修饰的切除机制和调控过程。
