Homology-dependent repair of DNA double-strand breaks (DSBs) by gene conversion involves short tracts of DNA synthesis and limited loss of heterozygosity (LOH). For DSBs that present only one end, repair occurs by invasion into a homologous sequence followed by replication to the end of the chromosome resulting in extensive LOH, a process called break-induced replication (BIR). We developed a BIR assay in Saccharomyces cerevisiae consisting of a plasmid with a telomere seeding sequence separated from sequence homologous to chromosome III by an I-SceI endonuclease recognition site. Following cleavage of the plasmid by I-SceI in vivo, de novo telomere synthesis occurs at one end of the vector, and the other end invades at the homologous sequence on chromosome III and initiates replication to the end of the chromosome to generate a stable chromosome fragment (CF). BIR was infrequent in wild-type cells due to degradation of the linearized vector. However, in the exo1Δ sgs1Δ mutant, which is defective in the 5′-3′ resection of DSBs, the frequency of BIR was increased by 39-fold. Extension of the invading end of the plasmid was detected by physical analysis two hours after induction of the I-SceI endonuclease in the wild-type exo1Δ, sgs1Δ, and exo1Δ sgs1Δ mutants, but fully repaired products were only visible in the exo1Δ sgs1Δ mutant. The inhibitory effect of resection was less in a plasmid-chromosome gene conversion assay, compared to BIR, and products were detected by physical assay in the wild-type strain. The rare chromosome rearrangements due to BIR template switching at repeated sequences were increased in the exo1Δ sgs1Δ mutant, suggesting that reduced resection can decrease the fidelity of homologous recombination.

The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.Homology-directed repair (HDR) allows cells to repair DNA double-strand breaks (DSBs) in an error-free way and is a key mechanism for maintaining genome integrity in all eukaryotes (11,48, 52). RAD52 is an essential gene for HDR in the yeast Saccharomyces cerevisiae (20,30,46). Identified as one member of an epistasis group of radiation-sensitive yeast mutants, rad52 strains are defective in HDR, the major DSB repair pathway in yeast. RAD52 is largely conserved throughout eukaryotes, and both the human and yeast Rad52 proteins stimulate the replacement of replication protein A with Rad51 on single-stranded DNA (4,29,39,45). Additionally, Rad52 promotes DNA annealing and strand exchange between a DNA duplex and complementary single-stranded DNA (14,21,27). Three-dimensional structures of the N terminus of human RAD52 (hRAD52) reveal a multimeric ring containing a positively charged surface groove that has been suggested to bind single-stranded DNA (15, 40). Relative to the N terminus of Rad52, the C terminus is poorly conserved between homologs. The C terminus of Rad52 is essential for its interaction with Rad51, a bacterial RecA homolog (26, 37). Consistent with its ability to bind both itself and other DNA repair proteins, Rad52 forms discrete nuclear foci both spontaneously and in response to DNA damage, and these DNA repair centers are the sites of DSB repair (23)(24)(25)32).Although yeast RAD52 plays a critical role in HDR, surprisingly, RAD52 knockout mice are resistant to DNA damage (35). However, it is likely that mammalian RAD52 is important for DNA repair, as its activities are conserved with yeast Rad52; in ad...