Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae

Abstract

Recombination plays a central role in the repair of broken chromosomes in all eukaryotes. We carried out a systematic study of mitotic recombination. Using several assays, we established the chronological sequence of events necessary to repair a single double-strand break. Once a chromosome is broken, yeast cells become immediately committed to recombinational repair. Recombination is completed within an hour and exhibits two kinetic gaps. By using this kinetic framework we also characterized the role played by several proteins in the recombinational process. In the absence of Rad52, the broken chromosome ends, both 5' and 3', are rapidly degraded. This is not due to the inability to recombine, since the 3' single-stranded DNA ends are stable in a strain lacking donor sequences. Rad57 is required for two consecutive strand exchange reactions. Surprisingly, we found that the Srs2 helicase also plays an early positive role in the recombination process.

FIG. 1.

(A) Three alternative models of DSB-initiated recombination resulting in gene conversion. (Panel 1) DSBR model. The DSBR model involves invasion of a homologous template by the two broken ends and formation of an intermediate containing two Holliday junctions. After mismatch repair, resolution leads to gene conversion. (Panel 2) One-ended SDSA model. A single broken end invades the homologous sequence and primes DNA synthesis. Newly synthesized DNA reanneals and ligates to the opposite broken arm. A second round of mismatch repair results in gene conversion. (Panel 3) Double-ended SDSA model (DE-SDSA). Invasion is carried out by the two broken arms, followed by DNA synthesis and annealing of the two newly synthesized DNA. Only one round of hDNA repair is necessary for gene conversion. B and R represent the BamHI and EcoRI restriction site polymorphisms, respectively. (B) Schematic representation of our experimental system. Open rectangles represent the ura3 alleles on chromosomes II and V. A black box represents the HOcs; a gray box depicts the inactive HOcs-inc flanked by the BamHI (B) and EcoRI (R) restriction sites. Transfer of the cells to galactose-containing medium results in gene conversion.

FIG. 2.

(A) Southern blot analysis of DNA extracted from MK203 at timely intervals. The DNA was digested with ClaI and probed with a fragment of chromosome V carrying the URA3 gene (hatched box) and with LEU2 sequences that served as a loading standard. (B) Quantitative PCR measures the relative amounts of intact chromosome V. Pictured below is a quantitative control reaction with primers complementary to a region on chromosome VIII. (C) Quantitation of Southern blot analysis. “Percent of broken chr. V” refers to the levels of the 2.8 and 1.2 kb fragments resulting from DSB formation. “Ratio of chr. V/chr. III” refers to the ratio between the 4-kb fragment representing unbroken chr. V sequences and the LEU2 band. hs, hour(s).

FIG. 3.

(A) Accumulation of ssDNA intermediates: results from nondenaturative slot blots with four RNA probes is plotted as a function of time. (B) Analysis of ssDNA resection in strain MK205. Southern blot analysis of DNA digested with ClaI and BglII are shown. Additional probes homologous to the LEU2 and TRP1 sequences were used as loading controls. hs, hour(s).

FIG. 4.

(A) Invasion and DNA synthesis assay. Primers adjacent to homology on chromosomes V and II amplify the polymerized invading intermediate. (B) hDNA repair is already maximal at the first time point at which the invasion-extension intermediate can be amplified. (C) Gene conversion assay. Equal amounts of PCR products obtained with primers a and b were digested with BamHI and subjected to gel electrophoresis. (D) Asymmetric repair during gene conversion. Equal amounts of PCR products obtained with primers b and d (upper panels) or primers a and c (lower panels) were digested with either EcoRI or BamHI. hs, hour(s).

FIG. 4.

(A) Invasion and DNA synthesis assay. Primers adjacent to homology on chromosomes V and II amplify the polymerized invading intermediate. (B) hDNA repair is already maximal at the first time point at which the invasion-extension intermediate can be amplified. (C) Gene conversion assay. Equal amounts of PCR products obtained with primers a and b were digested with BamHI and subjected to gel electrophoresis. (D) Asymmetric repair during gene conversion. Equal amounts of PCR products obtained with primers b and d (upper panels) or primers a and c (lower panels) were digested with either EcoRI or BamHI. hs, hour(s).

FIG. 5.

(A) Kinetics of gene conversion, as measured by the PCR gene conversion assay. (B) CCG. (C) Survival after DSB induction. hs, hour(s).

FIG. 6.

(A) ssDNA resection in strains MK203, MK203rad52, and MK205. (B) Quantitative PCR of remaining chromosome V in strains MK205 and MK203rad52. hs, hour(s).

FIG. 7.

(A) Southern blot analysis of DNA extracted from MK203rad57 at timely intervals. (B) Analysis of recombination intermediates in strain MK203rad57 PCR assays for intact chromosome V, invasion plus DNA synthesis, and gene conversion. hs, hour(s).

FIG. 8.

(A) Southern blot analysis of DNA extracted from MK203srs2 at timely intervals. The LEU2 fragment shown is derived from the srs2::LEU2 allele. (B) Analysis of recombination intermediates in strain MK203srs2 PCR assays for intact chromosome V, invasion plus DNA synthesis, and gene conversion. For the gene conversion analysis, equal amounts of PCR products obtained with primers a and b were digested with BamHI and subjected to gel electrophoresis. hs, hour(s).

FIG. 9.

Comparison of the kinetics of accumulation of the different intermediates in the recombination process. Symbols: □, DSB (Southern blot analysis); ◊, chromosome break (PCR); ○, ssDNA filament; ▵, invasion; ⊞, gene conversion. hs, hour(s).