title: Limiting the persistence of a chromosome break diminishes its mutagenic potential.
abstract: To characterize the repair pathways of chromosome double-strand breaks (DSBs), one approach involves monitoring the repair of site-specific DSBs generated by rare-cutting endonucleases, such as I-SceI. Using this method, we first describe the roles of Ercc1, Msh2, Nbs1, Xrcc4, and Brca1 in a set of distinct repair events. Subsequently, we considered that the outcome of such assays could be influenced by the persistent nature of I-SceI-induced DSBs, in that end-joining (EJ) products that restore the I-SceI site are prone to repeated cutting. To address this aspect of repair, we modified I-SceI-induced DSBs by co-expressing I-SceI with a non-processive 3' exonuclease, Trex2, which we predicted would cause partial degradation of I-SceI 3' overhangs. We find that Trex2 expression facilitates the formation of I-SceI-resistant EJ products, which reduces the potential for repeated cutting by I-SceI and, hence, limits the persistence of I-SceI-induced DSBs. Using this approach, we find that Trex2 expression causes a significant reduction in the frequency of repair pathways that result in substantial deletion mutations: EJ between distal ends of two tandem DSBs, single-strand annealing, and alternative-NHEJ. In contrast, Trex2 expression does not inhibit homology-directed repair. These results indicate that limiting the persistence of a DSB causes a reduction in the frequency of repair pathways that lead to significant genetic loss. Furthermore, we find that individual genetic factors play distinct roles during repair of non-cohesive DSB ends that are generated via co-expression of I-SceI with Trex2.
authors.affiliation: Department of Cancer Biology, Division of Radiation Biology, Beckman Research Institute of the City of Hope, Duarte, California, USA.
authors.forename: Nicole
authors.initials: N
authors.lastname: Bennardo
authors.forename: Amanda
authors.initials: A
authors.lastname: Gunn
authors.forename: Anita
authors.initials: A
authors.lastname: Cheng
authors.forename: Paul
authors.initials: P
authors.lastname: Hasty
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Stark
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D019457:Chromosome Breakage; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016296:Mutagenesis; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D005090:Exodeoxyribonucleases; C540589:Trex2 protein, mouse; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific