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Limiting the persistence of a chromosome break diminishes its mutagenic potential.

Authors :
Nicole Bennardo
Amanda Gunn
Anita Cheng
Paul Hasty
Jeremy M Stark
Source :
PLoS Genetics, Vol 5, Iss 10, p e1000683 (2009)
Publication Year :
2009
Publisher :
Public Library of Science (PLoS), 2009.

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.

Subjects

Subjects :
Genetics
QH426-470

Details

Language :
English
ISSN :
15537390 and 15537404
Volume :
5
Issue :
10
Database :
Directory of Open Access Journals
Journal :
PLoS Genetics
Publication Type :
Academic Journal
Accession number :
edsdoj.37f94376d0514eff8af9d0448fd73e77
Document Type :
article
Full Text :
https://doi.org/10.1371/journal.pgen.1000683