Methods to Study DNA End Resection I: Recombinant Protein Purification
Abstract 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.