Characterization of RAD51-independent break-induced replication that acts preferentially with short homologous sequences

Abstract

Repair of double-strand breaks by gene conversions between homologous sequences located on different Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences between the RAD51- and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the absence of telomerase.

FIG. 1.

Models of RAD51-dependent and RAD51-independent intraplasmid recombination between inverted repeats (thick lines). One end invades and primes new DNA synthesis (dashed lines). (a) In the presence of Rad51p, DSB repair occurs by gene conversion, which in mitosis is mostly not associated with crossovers. (b) In the absence of Rad51p, DSB repair occurs by BIR-SSA. Strand invasion sets up a replication fork in which both leading and lagging strand synthesis occurs. If synthesis goes to the very end of the linearized plasmid, repair can be completed by SSA, in which the ends are resected by 5′ to 3′ exonucleases. Alternative annealing between repeats yields equal numbers of crossover and noncrossover products. The position of the PstI cut site is changed in the crossover product.

FIG. 2.

Plasmid and chromosomal constructs. Open rectangles represent homologous sequences. Plasmids carry two inverted or direct repeats of MATa. The recipient copy containing an HO cleavage site was inserted between the EcoRI and HindIII sites, and the donor copy with a mutated HO cut site was inserted at the SmaI site of YCp50. (a) In one set of plasmids the donor sequence cannot be cleaved because of a single C to T substitution, designated MATa-inc. (b) Plasmids in which the donor and recipient MATa repeats are perfectly homologous, but in which the donor cannot be cleaved by HO because of a 20-bp insertion at the HO cut site. (c) Interplasmid recombination system: MATa and MATa-inc sequences are on different plasmids, marked with URA3 or LEU2. (d) The MATa sequence and the first 33 bp of the MATa sequence upstream of the HO cut site were inserted in the same orientation between the EcoRI and HindIII sites and between the HindIII and BamHI sites of YCp50, respectively. Here, DSB can only be repaired by SSA. (e) Interchromosomal ectopic recombination system: the MATa-inc sequence is located on chromosome III, and the MATa sequence marked by an adjacent hygromycin resistance gene (HYG) was inserted in the ARG5,6 locus on chromosome V.

FIG. 3.

DSB repair efficiency of two plasmids containing inverted repeats with intermediate lengths of homology (a) or short 33-bp inverted repeats (b). In both cases the DSB ends are perfectly homologous to those of the donor. The effect of deleting several genes important for recombination was determined. WT, wild type.

FIG. 4.

DSB repair efficiency for a set of plasmids carrying inverted MATa and MATa-inc repeats of various lengths. The effect of different homology lengths as well as the effects of deleting several genes involved in recombination is shown. DSB repair efficiency is plotted against the length of homology on each side of the DSB in a range of 25 to 70 bp (a) and in a range of 25 bp to 1 kb (b). WT, wild type.

FIG. 5.

The effect of homology length on the frequency of crossing over accompanying gene conversion. (a) Crossover frequencies were determined by densitometry of Southern blots for a set of strains with plasmids carrying inverted repeats of MATa and MATa-inc. Wild type (WT), rad59Δ, and rad51Δ mutants were analyzed. Crossover product bands are indicated by multiplication signs, and gene conversion without crossover product bands are indicated by equal signs. (b) The percentage of crossover products was plotted against the length of homology for the plasmids shown in panel a and a set of plasmids containing inverted repeats of MATα and MATα-inc.