Analysis of DNA double-strand break repair by nonhomologous end joining in cell-free extracts from mammalian cells.
Double-strand breaks (DSBs) in genomic DNA are induced by ionizing radiation or radiomimetic drugs, but they 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. NHEJ 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 hamster cell lines have yielded insights into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells have 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 that permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.
Pfeiffer
Petra
P
Institute of Genetics, University of Essen, Germany.
Feldmann
Elke
E
Odersky
Andrea
A
Kuhfittig-Kulle
Steffi
S
Goedecke
Wolfgang
W
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002457:Cell Extracts / Q000737:chemistry; D002474:Cell-Free System; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis / Q000737:chemistry; D004249:DNA Damage; D004260:DNA Repair; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic
D002457:Cell Extracts; D004247:DNA