Nucleic Acids Research Advance Access published online on August 1, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm521
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Molecular Biology |
Capture of linear fragments at a double-strand break in yeast
1Graduate School of Biomedical Sciences, University of Medicine & Dentistry of New Jersey and Rutgers University, Piscataway, NJ 08854, USA, 2Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel and 3Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA
*To whom correspondence should be addressed. Tel: +972-3-640-9031; Fax: +972-3-640-940; Email: martin{at}post.tau.ac.il
Received May 20, 2007. Revised June 20, 2007. Accepted June 21, 2007.
Double-strand breaks (DSBs) are dangerous chromosomal lesions that must be efficiently repaired in order to avoid loss of genetic information or cell death. In all organisms studied to date, two different mechanisms are used to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Previous studies have shown that during DSB repair, non-homologous exogenous DNA (also termed ‘filler DNA’) can be incorporated at the site of a DSB. We have created a genetic system in the yeast Saccharomyces cerevisiae to study the mechanism of fragment capture. Our yeast strains carry recognition sites for the HO endonuclease at a unique chromosomal site, and plasmids in which a LEU2 gene is flanked by HO cut sites. Upon induction of the HO endonuclease, a linear extrachromosomal fragment is generated in each cell and its incorporation at the chromosomal DSB site can be genetically monitored. Our results show that linear fragments are captured at the repaired DSB site at frequencies of 10–6 to 10–4 per plated cell depending on strain background and specific end sequences. The mechanism of fragment capture depends on the NHEJ machinery, but only partially on the homologous recombination proteins. More than one fragment can be used during repair, by a mechanism that relies on the annealing of small complementary sequences. We present a model to explain the basis for fragment capture.
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