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. 1997 Aug 19;94(17):9214-9.
doi: 10.1073/pnas.94.17.9214.

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination

N Sugawara  1 , F Pâques, M Colaiácovo, J E Haber
Affiliations

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination

N Sugawara et al. Proc Natl Acad Sci U S A. .

Abstract

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3' tail.

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Figures

Figure 1
Figure 1
Effect of MSH and RAD genes on HO endonuclease-induced gene conversion repair of a DSB. (A) Structure of the pFP122, pFP140, and pFP120 plasmids. (B) DSB repair efficiency in wild-type, rad1, msh2, msh3, msh2 rad1, and msh3 rad1 strains.
Figure 3
Figure 3
Requirement for Msh2, Msh3, and Rad1 in SSA. (A) SSA between identical 205-bp URA3 gene segments flanking an HO-induced DSB. Extensive 5′ to 3′ exonuclease digestion produces 3′ ended DNA tails in which complementary regions can anneal. Cleavage of the 3′-ended tails allows new DNA synthesis to fill in gaps and ligate the deletion. Diagnostic BglII fragments are indicated. (B) Southern blot of the time course of deletion formation in wild-type cells. BglII-digested DNA reveals HO cleavage and the formation of the deletion in the wild-type strain (tNS1379). Deletion formation is significantly reduced in msh2 (tNS1406), msh3 (tNS1390), and rad1 (tNS1429) strains. SSA was analyzed by probing with a HindIII–BamHI DNA fragment centromere proximal to the URA3 gene (8). (C) Southern blot of the time course of deletion formation on plasmid pNSU208 carrying 240 bp of directly repeated LacZ sequences in wild-type cells (tNS865), compared with msh3 (tRKY3112) and msh6 (tRKY3111) strains. DNA was digested with EcoRI. All strains were derivatives of tNR85 (22) and carried GAL::HO on a TRP1-marked plasmid, pFH800 (27).
Figure 4
Figure 4
Effect of PMS1 and MLH1 on SSA. An experiment similar to that of Fig. 3 was performed except that the 205-bp repeats flanking the DSB differed by seven heterologies (see Materials and Methods). Strains included wild type (tNS1357), mlh1 (tNS1396), pms1 (tNS1394), and msh2 (tNS1359).
Figure 5
Figure 5
Effect of length of the annealed region on requirements for RAD and MSH genes during SSA. Experiments identical to those shown in Fig. 3 were carried out with SSA substrates with different lengths of homology flanking the HO-induced DSB. Strains were identical to those used in Fig. 3, whose data are plotted here for the experiment with a length of 205 bp. The lengths of the nonhomologous tails to be excised were the same in all cases. Data for rad1, msh2, and msh3 were normalized to the wild-type value at each point.
Figure 2
Figure 2
Kinetics of HO-induced crossing-over. (A) When pFP122 undergoes HO cleavage, gene conversions both with and without crossing-over arise. The crossover products can be distinguished on Southern blots, probed with LacZ sequences, by the sizes of PstI-restriction fragments homologous to LacZ (1, 10). HO-induced re-combination in wild-type (B), msh2 (C), and rad1 (D) strains exhibit the appearance of two crossover products one hr after HO cleavage of the 5.2-kb PstI LacZ fragment. One of the two gene conversion products comigrates with the 5.2-kb parental fragment. However, in these strains carrying an integrated ADE3::HO gene, HO cleaves essentially all of the plasmid targets (data not shown), so that virtually all of the 5.2-kb band remaining at the end of the experiment represents gene conversions without crossing-over and not the original parental fragment. The sum of the intensities of the 4.3- and 8.5-kb crossover bands divided by the sum of the crossover bands and the 5.2- and 8.3-kb noncrossover bands, measured 24 hr after HO induction, indicates the proportion of gene conversions associated with crossing-over.
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