Analysis of DNA double-strand break (DSB) repair in mammalian cells.
DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.
Seluanov
Andrei
A
Department of Biology, University of Rochester.
Mao
Zhiyong
Z
Gorbunova
Vera
V
D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D005347:Fibroblasts / Q000502:physiology; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D015202:Protein Engineering / Q000379:methods; D011995:Recombination, Genetic; D014162:Transfection
D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins