In vitro rejoining of double-strand breaks in genomic DNA.
Recent genetic and biochemical studies have provided important insights into the mechanism of nonhomologous end-joining (NHEJ) in higher eukaryotes, and have helped to characterize several components including DNA-PKcs, Ku, DNA ligase IV, and XRCC4. There is evidence, however, that additional factors involved in NHEJ remain to be characterized. The biochemical characterization of NHEJ in higher eukaryotes has benefited significantly from in vitro plasmid-based end-joining assays. However, because of differences in the organization and sequence of genomic and plasmid DNA, and because multiple pathways of NHEJ are operational, it is possible that different factors are preferred for the rejoining of double-strand breaks (DSBs) induced in plasmid vs genomic DNA organized in chromatin. Here, we describe an in vitro assay that allows the study of DSB rejoining in genomic DNA. The assay utilizes as a substrate DSBs induced by various means in genomic DNA prepared from agarose-embedded cells after appropriate lysis. Two extremes in terms of state of DNA organization are described: "naked" DNA and DNA organized in chromatin. Here, we describe the protocols developed to carry out and analyze these in vitro reactions, including procedures for preparation of cell extract and the preparation of the substrate DNA ("naked" DNA or nuclei).
Iliakis
George
G
Institute of Medical Radiation Biology, Medical School, University of Duisburg-Essen, Essen, Germany.
Cheong
Nge
N