Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication

Abstract

Toxic recombination events are detected in vegetative Saccharomyces cerevisiae cells through negative growth interactions between certain combinations of mutations. For example, mutations affecting both the Srs2 and Sgs1 helicases result in extremely poor growth, a phenotype suppressed by mutations in genes that govern early stages of recombination. Here, we identify a similar interaction involving double mutations affecting Sgs1 or Top3 and Mus81 or Mms4. We also find that the primary DNA structures that initiate these toxic recombination events cannot be double-strand breaks and thus are likely to be single-stranded DNA. We interpret our results in the context of the idea that replication stalling leaves single-stranded DNA, which can then be processed by two competing mechanisms: recombination and nonrecombination gap-filling. Functions involved in preventing toxic recombination would either avoid replicative defects or act on recombination intermediates. Our results suggest that Srs2 channels recombination intermediates back into the gap-filling route, whereas Sgs1Top3 and Mus81Mms4 are involved in recombination andor in replication to allow replication restart.

Fig 1.

Inactivation of recombination suppresses the synthetic lethality of mus81 sgs1 and mms4 sgs1 mutants. Tetrads from diploids heterozygous for the three mutations are indicated underneath the figures. The single “rad ” segregant clones are indicated by squares, the sgs1 mus81 and sgs1 mms4 double-mutant clones by circles, and the triple mutants by diamonds.

Fig 2.

Pathways involved in replication recovery, in wild-type and mutant cells. On replication arrest, ssDNA is processed either by a nonrecombinogenic gap-filling mechanism or by HR. In model 1, Sgs1 acts in replication to prevent the formation of ssDNA. In model 2, Sgs1 acts in one of the recombination subpathways. “(m)” indicates the channeling of the intermediate in the corresponding single mutant (see text for explanations).

Fig 3.

Molecular model for the functions of Srs2, Mus81, and Sgs1 during recombinational repair of replicative defects. Recombination initiated from a single-stranded gap leads after strand invasion and DNA synthesis to intermediates that can be metabolized through two pathways leading to a double HJ structure (A) or, on strand displacement, to a 3′ tail protruding from the initially gapped molecule (B). This scheme applies to the second model shown in Fig. 2 (see text for explanations). (Lower) The fate of the intermediate structures in the rad54, mus81, or sgs1 mutants.