Cells Expressing Murine RAD52 Splice Variants Favor Sister Chromatid Repair

Abstract

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...

Interaction with RPA Is Necessary for Rad52 Repair Center Formation and for Its Mediator Activity

“…In fact, for each of the other rad52 truncation mutant strains, the percentage of cells with foci is reduced more than 10-fold compared with wild type, irrespective of the cell cycle stage investigated. In agreement with this, it has recently been reported that a C-terminal Rad52 truncation terminating at residue 284 does not form foci (38).…”

The Rad52 Homologs Rad22 and Rti1 ofSchizosaccharomyces pombeAre Not Essential for Meiotic Interhomolog Recombination, but Are Required for Meiotic Intrachromosomal Recombination and Mating-Type-Related DNA Repair

“…The identification of intrachromosomal recombination as the major meiotic function of Rad22 and Rti1 is in line with the observation that meiotic Holliday junctions in S. pombe involve sister chromatids four times more often than homologs (Cromie et al 2006). A recent study has shown that truncated RAD52 polypeptides mimicking murine splicing variants are able to favor sister chromatid recombination in S. cerevisiae (Thorpe et al 2006). In addition, we estimate that in a cell the overall number of HR events including repair involving sister chromatids largely exceeds the average observed number of 20 Rad51 foci (Figure 3).…”

Identification of a Second DNA Binding Site in the Human Rad52 Protein

“…These observations suggest that the functions exhibited by the second DNA binding site of Rad52 are shared between Rad52 and Rad59 and are an important component in the Rad52-mediated DSB repair process. Although a homolog of Rad59 has not been found in higher eukaryotes, a functional counterpart, perhaps the splicing variant of Rad52 (36,37), may play a similar role to that of Rad59.…”