Assays for nonhomologous end joining in extracts.
In mammalian cells, nonhomologous end-joining (NHEJ) repairs DNA double strand breaks created by ionizing radiation and V(D)J recombination. Using human whole cell extracts prepared by the method of Baumann and West (1998), we have described a cell-free system for NHEJ that joins both compatible and noncompatible DNA ends (Budman and Chu, 2005). To measure joining efficiency and assess the processing of DNA ends, we developed a quantitative polymerase chain reaction assay for the joining of two specific DNA ends. The in vitro NHEJ reaction recapitulates key features of NHEJ observed in vivo: end joining is dependent on DNA-PK and XRCC4/Ligase4, and noncompatible ends are processed by polymerase and nuclease activities that often stabilize the alignment of opposing ends by base pairing. This chapter describes methods for preparing whole cell extracts and for studying the NHEJ reaction in vitro.
Budman
Joe
J
Department of Medicine, Stanford University School of Medicine, California, USA.
Chu
Gilbert
G
D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D011995:Recombination, Genetic; D045567:VDJ Recombinases / Q000378:metabolism
D004268:DNA-Binding Proteins; C101467:XRCC4 protein, human; D051747:DNA-Activated Protein Kinase; D045567:VDJ Recombinases