Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair.
DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast andDrosophilaTo better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells.
Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, North Carolina 27599.
Grzegorz
G
Zapotoczny
Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, North Carolina 27599 sekelsky@unc.edu.|Department of Biology, University of North Carolina at Chapel Hill, North Carolina 27599.|Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, North Carolina 27599.
Jeff
0000-0002-4424-677X
J
Sekelsky
D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D003434:Crossing Over, Genetic; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics*; D055785:Gene Knockdown Techniques; D006801:Humans; D008957:Models, Genetic; D011995:Recombination, Genetic / Q000235:genetics
crossing over; double-strand break repair; synthesis-dependent strand annealing