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.
Subject(s)
Cell Culture Techniques/methods , DNA Breaks, Double-Stranded , Enzyme Assays/methods , Recombinant Proteins/isolation & purification , Recombinational DNA Repair , Acid Anhydride Hydrolases , Animals , Baculoviridae/genetics , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Cell Culture Techniques/instrumentation , Cell Cycle Proteins/isolation & purification , Cell Cycle Proteins/metabolism , DNA Helicases/isolation & purification , DNA Helicases/metabolism , DNA Repair Enzymes/isolation & purification , DNA Repair Enzymes/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases , Enzyme Assays/instrumentation , Humans , MRE11 Homologue Protein/isolation & purification , MRE11 Homologue Protein/metabolism , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , RecQ Helicases/isolation & purification , RecQ Helicases/metabolism , Recombinant Proteins/metabolism , Replication Protein A/isolation & purification , Replication Protein A/metabolism , Sf9 Cells , Spodoptera , Transfection/methods , Werner Syndrome Helicase/isolation & purification , Werner Syndrome Helicase/metabolismABSTRACT
DNA end resection initiates the largely accurate repair of DNA double-strand breaks (DSBs) by homologous recombination. Specifically, recombination requires the formation of 3' overhangs at DSB sites, which is carried out by nucleases that specifically degrade 5'-terminated DNA. In most cases, DNA end resection is a two-step process, comprising of initial short-range followed by more processive long-range resection. In this chapter, we describe selected assays that reconstitute both the short- and long-range pathways. First, we define methods to study the exonuclease and endonuclease activities of the MRE11-RAD50-NBS1 (MRN) complex in conjunction with phosphorylated cofactor CtIP. This reaction is particularly important to initiate processing of DNA breaks and to recruit components belonging to the subsequent long-range pathway. Next, we describe assays that reconstitute the concerted reactions of Bloom (BLM) or Werner (WRN) helicases that function together with the DNA2 nuclease-helicase, and which are as a complex capable to resect DNA of kilobases in length. The reconstituted reactions allow us to understand how the resection pathways function at the molecular level. The assays will be invaluable to define regulatory mechanisms and to identify inhibitory compounds, which may be valuable in cancer therapy.
