Rad51 Protein Controls Rad52-mediated DNA Annealing

Abstract

In Saccharomyces cerevisiae, Rad52 protein plays an essential role in the repair of DNA double-stranded breaks (DSBs). Rad52 and its orthologs possess the unique capacity to anneal single-stranded DNA (ssDNA) complexed with its cognate ssDNA-binding protein, RPA. This annealing activity is used in multiple mechanisms of DSB repair: single-stranded annealing, synthesis-dependent strand annealing, and crossover formation. Here we report that the S. cerevisiae DNA strand exchange protein, Rad51, prevents Rad52-mediated annealing of complementary ssDNA. Efficient inhibition is ATP-dependent and involves a specific interaction between Rad51 and Rad52. Free Rad51 can limit DNA annealing by Rad52, but the Rad51 nucleoprotein filament is even more effective. We also discovered that the budding yeast Rad52 paralog, Rad59 protein, partially restores Rad52-dependent DNA annealing in the presence of Rad51, suggesting that Rad52 and Rad59 function coordinately to enhance recombinational DNA repair either by directing the processed DSBs to repair by DNA strand annealing or by promoting second end capture to form a double Holliday junction. This regulation of Rad52-mediated annealing suggests a control function for Rad51 in deciding the recombination path taken for a processed DNA break; the ssDNA can be directed to either Rad51-mediated DNA strand invasion or to Rad52-mediated DNA annealing. This channeling determines the nature of the subsequent repair process and is consistent with the observed competition between these pathways in vivo.In Saccharomyces cerevisiae, the repair of DNA doublestranded breaks (DSBs) 3 is accomplished primarily by homologous recombination. Genes from the RAD52 epistasis group, including RAD50, RAD51, RAD52, RAD54, RAD55-57, RAD59, MRE11, XRS2, and RFA1, are responsible for this recombination-dependent DSB repair (1, 2). To repair a DSB, the DNA end is first processed to produce a 3Ј single-stranded tailed duplex DNA. The ssDNA is then channeled into one of the many recombinational pathways, which can be further categorized into RAD51-dependent and -independent pathways.RAD51-dependent recombination requires functions of RAD51, RAD52, RAD54, RAD55, RAD57, and RFA1 for efficient DNA repair (1). The central step of this pathway involves DNA strand invasion of homologous duplex DNA by the processed DSB complexed with Rad51 protein (3). DNA replication from the invading 3Ј-end replaces the genetic information missing from the broken dsDNA, and subsequent DNA pairing and resolution steps restore DNA integrity.RAD51-independent recombination requires RAD52 (4) and is enhanced by RAD59 (5-7). In addition to the recombination genes MRE11, RAD50, XRS2, RAD52, and RAD59 (6,8), this pathway also depends on MSH2, MSH3 (6, 9), RAD1, and RAD10 (10, 11). This RAD51-independent recombination is most easily assayed as DSB repair occurring between directly repeated sequences, by a mechanism termed single-stranded annealing (SSA) (8, 12). As implied, the central step of SSA is annealing between complementary singl...

Regulation of Recombination and Genomic Maintenance

“…Dissociation of Rad51 from the invading end avoids HR (antirecombination), whereas dissociation of Rad51 from the second, noninvading end could enable strand annealing during SDSA (anticrossover). This interpretation is consistent with genetic and biochemical evidence showing that Rad51 impedes ssDNA annealing mediated by Rad52 (Dupaigne et al 2008;Wu et al 2008;Carter et al 2009;Mitchel et al 2013;Miura et al 2013). RTEL1, a DNA helicase found in metazoans but not fungi, displays genetic properties reminiscent of yeast srs2 mutants including DNA damage sensitivity, hyperrecombination, and colethality with mutations in SGS1/BLM (Barber et al 2008).…”

Leaping forks at inverted repeats: Figure 1.

“…However, if different pathways contribute to the outcome, the individual role of the distinct factors involved may be difficult to establish genetically. In vitro, Rad51 inhibits the SSA activity of Rad52 (Wu et al 2008). The observation that Srs2 (in which Rad51 is somewhat up-regulated by failure to disrupt Rad51 filaments) is required for the fusion events observed may mirror a possible role for Rad52 in this process.…”

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

“…The Rad51 filament continues to interact with Rad52 via protein -protein interactions (Wu et al 2008). The Rad51 filament now promotes pairing and invasion of the first end to its homologous sequence on the sister chromatid (Fig.…”

“…3) (Davis and Symington 2003). The result is second-end capture (Davis and Symington 2001;Wu et al 2006Wu et al , 2008 and the completion of DSBR by the NCO mechanism. In summary, sequential handoffs and coordinated activities between DNA-pairing and annealing proteins and their partners explain how SDSA effects the repair of DNA DSBs while minimizing risks associated with COs and LOH.…”

“…Third, Rad51 in the postsynaptic filament interacts locally with Rad52 and suppresses its annealing activity (Fig. 3) (Wu et al 2008). The latter effect suppresses the formation of dHJs and, thus, avoids accidental formation of genotoxic COs.…”