Methods to Study DNA End Resection II: Biochemical Reconstitution Assays

• Published in: Methods in enzymology Vol. 600; p. 67
• Main Authors: Pinto, Cosimo, Anand, Roopesh, Cejka, Petr
• Format: Journal Article
• Language: English
• Published: United States 2018
• Subjects: Acid Anhydride Hydrolases; Animals; Baculoviridae - genetics; Buffers; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Cell Culture Techniques - instrumentation; Cell Culture Techniques - methods; Cell Cycle Proteins - isolation & purification; Cell Cycle Proteins - metabolism; DNA Breaks, Double-Stranded; 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; Electrophoresis, Polyacrylamide Gel - instrumentation; Electrophoresis, Polyacrylamide Gel - methods; Endodeoxyribonucleases; Enzyme Assays - instrumentation; Enzyme Assays - methods; Humans; MRE11 Homologue Protein - isolation & purification; MRE11 Homologue Protein - metabolism; Nuclear Proteins - isolation & purification; Nuclear Proteins - metabolism; Oligonucleotides - metabolism; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombinational DNA Repair; RecQ Helicases - isolation & purification; RecQ Helicases - metabolism; Replication Protein A - isolation & purification; Replication Protein A - metabolism; Sf9 Cells; Spodoptera; Transfection - methods; Werner Syndrome Helicase - isolation & purification; Werner Syndrome Helicase - metabolism; Helicase; In vitro assay; Mre11; Homologous recombination; Bloom; Biochemistry; Dna2; Nuclease; DNA end resection; Werner
• ISSN: 1557-7988
• DOI: 10.1016/bs.mie.2017.11.009

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.