DNA Annealing Mediated by Rad52 and Rad59 Proteins

Abstract

In the budding yeast Saccharomyces cerevisiae, the RAD52 gene is essential for all homologous recombination events and its homologue, the RAD59 gene, is important for those that occur independently of RAD51. Both Rad52 and Rad59 proteins can anneal complementary single-stranded (ss) DNA. We quantitatively examined the ssDNA annealing activity of Rad52 and Rad59 proteins and found significant differences in their biochemical properties. First, and most importantly, they differ in their ability to anneal ssDNA that is complexed with replication protein A (RPA). Rad52 can anneal an RPA-ssDNA complex, but Rad59 cannot. Second, Rad59-promoted DNA annealing follows first-order reaction kinetics, whereas Rad52-promoted annealing follows second-order reaction kinetics. Last, Rad59 enhances Rad52-mediated DNA annealing at increased NaCl concentrations, both in the absence and presence of RPA. These results suggest that Rad59 performs different functions in the recombination process, and should be more accurately viewed as a Rad52 paralogue.Repair of DNA double-strand breaks (DSBs) 3 is a crucial process needed to maintain cell viability and genomic integrity. An unrepaired DSB can result in growth arrest, loss of genetic information, or cell death (1). In Saccharomyces cerevisiae, genes in the RAD52 epistasis group are responsible for homologous recombination-dependent DSB repair (2-4). To repair a DSB, the DNA end is first processed to reveal a 3Ј single-stranded (ssDNA) tail, which is subsequently channeled into one of the recombinational pathways. The canonical gene conversion pathway is an evolutionarily conserved process carried out by the DNA strand exchange protein, Rad51 (5). Genes that are important for the gene conversion pathways are RAD51, RAD52, RAD54, RAD55, RAD57, and RFA1, which collectively define the RAD51-dependent recombination pathway (2, 4).In addition to the RAD51-dependent recombination, alternative repair pathways exist that are independent of RAD51 function. These pathways have different but overlapping genetic requirements from the classical gene conversion pathway (1, 6 -8). In S. cerevisiae, the major RAD51-independent pathway is single-stranded annealing (SSA). The SSA pathway does not involve Rad51-mediated invasion of dsDNA and the subsequent strand exchange steps. Instead, it occurs by annealing of complementary ssDNA on either side of the DSB, followed by removal of heterologous tails and ligation of the nicks (2). In addition to the recombination genes MRE11, RAD50, XRS2, RAD52, and RAD59 (6, 7), the SSA pathway also depends on the MSH2, MSH3 (7, 9), RAD1 and RAD10 (9, 10) genes. SSA in yeast is highly effective, accounting for 35-80% of DSB repair events when a DSB occurs between two direct repeats (2, 6, 11).The RAD52 gene encodes a 471-residue protein that is required for all homologous recombination events in S. cerevisiae (2, 4). The ϳ200-residue N terminus of Rad52 protein is highly conserved in most eukaryotic species; whereas the C terminus confers species-specific interaction...

Mgm101 Is a Rad52-related Protein Required for Mitochondrial DNA Recombination

“…Yeast cells also express Rad59, a Rad52 paralogue that lacks the C-terminal Rad51-interacting domain (40). Rad59 functionally overlaps with Rad52 in DSB repair, but it also appears to have unique functions, including stimulation of Rad52-mediated single strand annealing (41)(42)(43).…”

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

“…Rad59 has its own intrinsic annealing activity that is kinetically distinct from Rad52 (Wu et al 2006). Unlike Rad52, the annealing activity of Rad59 is not stimulated by RPA (Petukhova et al 1999;Wu et al 2006). Rad59 also lacks recombination mediator activity, but it is biologically important in recombination pathways that do not require Rad51, such as SSA (Sugawara et al 2000;Pannunzio et al 2008Pannunzio et al , 2010.…”

“…This mechanism appears to be conserved as both RecO and UvsY show similar interactions with cognate SSB-ssDNA complexes (Sweezy and Morrical 1999;Ryzhikov et al 2011;Ryzhikov and Korolev 2012). The annealing activity of S. cerevisiae Rad52 protein is stimulated by Rad59, a paralog of Rad52 (Davis and Symington 2001;Wu et al 2006). Rad59 has its own intrinsic annealing activity that is kinetically distinct from Rad52 (Wu et al 2006).…”

“…The annealing activity of S. cerevisiae Rad52 protein is stimulated by Rad59, a paralog of Rad52 (Davis and Symington 2001;Wu et al 2006). Rad59 has its own intrinsic annealing activity that is kinetically distinct from Rad52 (Wu et al 2006). Unlike Rad52, the annealing activity of Rad59 is not stimulated by RPA (Petukhova et al 1999;Wu et al 2006).…”

“…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.…”