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Molecular and Cellular Biology, July 2000, p. 5300-5309, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
DNA Length Dependence of the Single-Strand Annealing Pathway
and the Role of Saccharomyces cerevisiae RAD59 in
Double-Strand Break Repair
Neal
Sugawara,
Grzegorz
Ira, and
James E.
Haber*
Rosenstiel Center and Department of Biology,
Brandeis University, Waltham, Massachusetts 02454-9110
Received 17 November 1999/Returned for modification 16 December
1999/Accepted 26 April 2000
A DNA double-strand break (DSB) created by the HO endonuclease in
Saccharomyces cerevisiae will stimulate recombination
between flanking repeats by the single-strand annealing (SSA) pathway, producing a deletion. Previously the efficiency of SSA, using homologous sequences of different lengths, was measured in competition with that of a larger repeat further from the DSB, which ensured that
nearly all cells would survive the DSB if the smaller region was not
used (N. Sugawara and J. E. Haber, Mol. Cell. Biol. 12:563-575, 1992). Without competition, the efficiency with which homologous segments of 63 to 205 bp engaged in SSA was significantly increased. A
sequence as small as 29 bp was used 0.2% of the time, and homology dependence was approximately linear up to 415 bp, at which size almost
all cells survived. A mutant with a deletion of RAD59, a
homologue of RAD52, was defective for SSA, especially when
the homologous-sequence length was short; however, even with 1.17-kb substrates, SSA was reduced fourfold. DSB-induced gene conversion also
showed a partial dependence on Rad59p, again being greatest when the
homologous-sequence length was short. We found that Rad59p plays a role
in removing nonhomologous sequences from the ends of single-stranded
DNA when it invades a homologous DNA template, in a manner similar to
that previously seen with srs2 mutants.
rad59 affected DSB-induced gene conversion differently
from msh3 and msh2, which are also defective in
removing nonhomologous ends in both DSB-induced gene conversion and
SSA. A msh3 rad59 double mutant was more severely defective
in SSA than either single mutant.
*
Corresponding author. Mailing address: Rosenstiel
Center and Department of Biology, Brandeis University, Waltham, MA
02454-9110. Phone: (781) 736-2462. Fax: (781) 736-2405. E-mail:
haber{at}brandeis.edu.
Molecular and Cellular Biology, July 2000, p. 5300-5309, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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