Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins Msh2 and Msh6 but not Pms1

Abstract

Recombination between moderately divergent DNA sequences is impaired compared with identical sequences. In yeast, an HO endonuclease-induced double-strand break can be repaired by single-strand annealing (SSA) between flanking homologous sequences. A 3% sequence divergence between 205-bp sequences flanking the double-strand break caused a 6-fold reduction in repair compared with identical sequences. This reduction in heteroduplex rejection was suppressed in a mismatch repair-defective msh6 Delta strain and partially suppressed in an msh2 separation-of-function mutant. In mlh1 Delta strains, heteroduplex rejection was greater than in msh6 Delta strains but less than in wild type. Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1 Delta mlh2 Delta mlh3 Delta triple mutant resembled mlh1 Delta. However, correction of the mismatches within heteroduplex SSA intermediates required PMS1 and MLH1 to the same extent as MSH2 and MSH6. An SSA competition assay in which either diverged or identical repeats can be used for repair showed that heteroduplex DNA is likely to be unwound rather than degraded. This conclusion is supported by the finding that deleting the SGS1 helicase also suppressed heteroduplex rejection.

Fig. 1.

SSA by using a homeologous substrate. (A) An in vivo DSB created between two repeated sequences initiates the 5′ to 3′ resection of one DNA strand, creating 3′ single-strand DNA tails. Annealing of single-strand DNA at complementary sequences creates an intermediate with mismatched base pairs. Nonhomologous tails are removed by Rad1p–Rad10p endonuclease with the assistance of Msh2p–Msh3p, and the gaps are filled in and ligated. If mismatches are not corrected by mismatch repair, progeny containing both genotypes (sectored colony) will result. (B) SSA substrates contain an HO cut site flanked by two or three 205-bp sequences derived from URA3. Black boxes indicate the repeated sequences.

Fig. 2.

Southern hybridization analysis of SSA. (A) HO endonuclease cleaves at its recognition site between 205-bp repeats of ura3 sequence (solid boxes) leading to a deletion. The ura3 sequence on the left consists of either the ura3-A or ura3-FL100 allele, whereas the right sequence contains ura3-A. The probe used for Southern blotting is a HindIII–BamHI fragment downstream of URA3. (B) DNA was extracted and digested with BglII from wild-type strains, tNS1379 and tNS1357 (A-A and F-A respectively), and the following derivatives of tNS1357: msh6Δ (tNS1600), msh2-R730W (tNS1826), sgs1Δ (EAY994), pms1Δ (tNS1394), mlh1Δ (tNS1396), mlh2Δ (tNS1916), mlh3Δ (tNS1909), exo1Δ (tNS1678), and srs2Δ (tNS1631). Southern blots show the uncut band before induction, the HO-cleaved band (4.8 kb) at 0.5 h, and the product band (5.5 kb) after 5 h of induction.

Fig. 3.

A competition assay to examine the mechanism of heteroduplex rejection. (A) A DSB was created so that the URA3 sequence to the right of the break can anneal with one of two 205-bp segments to the left to form a small or a large deletion. The three repeats are either all identical A sequences (A-A-A) or the middle repeat contains the 3% mismatched F sequence (A-F-A). (B) Southern blots (as described in Fig. 2) show that nearly all of the deletions in the A-F-A substrate (EAY1139) are to the distal, perfectly matched partner, in contrast to the A-A-A substrate in the wild-type (EAY1137), msh6Δ A-F-A, or sgs1Δ A-F-A strains. (C) Densitometric analysis of the kinetics of forming large (▪) and small (•) deletions in wild-type and msh6Δ A-F-A strains and large (□) and small (○) deletions in a wild-type A-A-A strain. (D) Wild-type (tNS1357) and msh6Δ (tNS1600) F-A strains were induced and DNA was extracted at the time points shown. SSA product was formed with the same kinetics when assayed by PCR with primers flanking the repeats. Reverse images are shown. Quantities of genomic DNA were adjusted so that equal amounts of final SSA product were formed by using DNA from wild-type and msh6Δ strains.