Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast

Abstract

Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Delta cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange.

Figure 1. Ectopic Gene Conversion Assay

(A) The experimental system to study ectopic gene conversion. A galactose inducible HO endonuclease generates a DSB within the 2 kb MATa sequence (marked by hygromycin resistance gene, HPH1) inserted at ARG5,6 on chromosome V. The homologous MATa-inc region on chromosome III is used as a donor for gene conversion. Both HML and HMR are deleted. Crossover and noncrossover products have different restriction fragment sizes and can be quantified on Southern blots. (B) Southern blot analysis of the proportion of gene conversions with and without crossover in strains lacking Srs2 and/or Sgs1. (C) Viability of srs2Δ, sgs1Δ, and rad59Δ cells after induction of a DSB.

Figure 2. srs2Δ and sgs1Δ Cells Exhibit Increased Levels of Crossovers

(A) Percentage of crossovers and noncrossovers among all cells that induced the DSB in the absence of DNA helicases Sgs1 and Srs2 or topoisomerase Top3. (B) Level of crossovers among the cells that successfully repaired the DSB in the absence of Sgs1 and Srs2 or Top3 and in cells arrested in G2/M with nocodazole.

Figure 3. Srs2 Suppresses Crossovers in Allelic Recombination

(A) Allelic recombination assay, crossover frequency is measured as the frequency of sectored Thr4^+/− colonies. (B) Viability and crossover frequency in WT and srs2Δ cells. (C) Comparison of allelic and ectopic recombinational repair kinetics.

Figure 4. Kinetics of Ectopic DSB Repair

Crossovers and noncrossovers appear with different kinetics in wild-type and sgs1Δ but not in srs2Δ. The total amount of product at 8 hr was 31 ± 5% in srs2Δ strain and 77 ± 18% in sgs1Δ relative to wild-type cells.

Figure 5. Suppression of the Elevated Level of Crossing-Over in sgs1Δ and srs2Δ Strains by Overexpression of SRS2 or SGS1

Genes were transcribed from their normal promoters unless otherwise specified.

Figure 6. Sgs1p Prevents and Srs2 Facilitates Recombination between Short Homologous Sequences

(A) In a plasmid used to study recombination, HO generates two 33 bp-long homologous DSB ends that can recombine with MATa-inc donor sequences situated in the opposite orientation. (B) Effect of different mutations on the efficiency of DSB repair in the plasmid shown in (A).

Figure 7. Model of Sgs1- and Srs2-Dependent Crossover Suppression

(A) Srs2 promotes the noncrossover SDSA pathway. (B) A HJ resolvase cuts double Holliday junctions to give crossovers and noncrossovers. (C) Sgs1 acts together with Top3 to remove double Holliday junctions so that gene conversions will be recovered as noncrossovers.