TY - JOUR
T1 - Capture of linear fragments at a double-strand break in yeast
AU - Haviv-Chesner, Anat
AU - Kobayashi, Yoshifumi
AU - Gabriel, Abram
AU - Kupiec, Martin
N1 - Funding Information:
This work was supported from grants by the Israeli Science Foundation and the Israeli Ministry of Health to M.K. and from National Institute of General Medical Sciences, the American Cancer Society and the New Jersey Commission for Cancer Research to A.G. We thank all members of the Kupiec and Gabriel laboratory for encouragement and comments on the manuscript. Funding to pay the Open Access publication charges for this article was provided by the Israeli Science Foundation.
PY - 2007/8
Y1 - 2007/8
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=34548585248&partnerID=8YFLogxK
U2 - 10.1093/nar/gkm521
DO - 10.1093/nar/gkm521
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AN - SCOPUS:34548585248
SN - 0305-1048
VL - 35
SP - 5192
EP - 5202
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 15
ER -