title: Analysis of double-strand break repair by nonhomologous DNA end joining in cell-free extracts from mammalian cells.
abstract: Double-strand breaks (DSB) in genomic DNA are induced by ionizing radiation or radiomimetic drugs but also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair which is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient cell lines yielded insight into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay which permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.
authors.affiliation: Institute of Genetics, University of Essen, Essen, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Pfeiffer
authors.forename: Andrea
authors.initials: A
authors.lastname: Odersky
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Goedecke
authors.forename: Steffi
authors.initials: S
authors.lastname: Kuhfittig-Kulle
mesh_terms: D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts / Q000235:genetics; D002460:Cell Line; D002474:Cell-Free System; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli; D006801:Humans; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics
chemical_list: D002457:Cell Extracts