Role of the Rad52 Amino-terminal DNA Binding Activity in DNA Strand Capture in Homologous Recombination

Abstract

Saccharomyces cerevisiae Rad52 protein promotes homologous recombination by nucleating the Rad51 recombinase onto replication protein A-coated single-stranded DNA strands and also by directly annealing such strands. We show that the purified rad52-R70A mutant protein, with a compromised aminoterminal DNA binding domain, is capable of Rad51 delivery to DNA but is deficient in DNA annealing. Results from chromatin immunoprecipitation experiments find that rad52-R70A associates with DNA double-strand breaks and promotes recruitment of Rad51 as efficiently as wild-type Rad52. Analysis of gene conversion intermediates reveals that rad52-R70A cells can mediate DNA strand invasion but are unable to complete the recombination event. These results provide evidence that DNA binding by the evolutionarily conserved amino terminus of Rad52 is needed for the capture of the second DNA end during homologous recombination. Homologous recombination (HR)2 represents an error-free pathway for the elimination of DNA breaks and DNA interstrand cross-links from chromosomes, and it also provides a means for the restart of stalled or damaged DNA replication forks. Moreover, HR plays a crucial role in the proper segregation of homologous chromosomes in meiosis I (for review, see Refs. 1 and 2). Accordingly, a deficiency in HR or its deregulation leads to meiotic aneuploidy, genomic instability, and the tumor phenotype (for review, see Refs. 1-3).HR is often initiated via the formation of DNA double-strand breaks (DSBs). After DSB formation, the ends of the break are nucleolytically processed to yield single-stranded DNA (ssDNA) tails (for review, see Ref. 4). The ubiquitous singlestrand DNA-binding protein RPA first decorates the ssDNA. RPA is subsequently replaced by the Rad51 recombinase to form a right-handed helical polymer, referred to as the presynaptic filament. This nucleoprotein complex conducts a search for a homologous DNA sequence and invades the latter to form a nascent DNA joint called the displacement loop, or D-loop (for review, see Ref. 5). After invasion, the 3Ј end of the invading strand primes DNA synthesis, which serves to replace the genetic information lost during the end-processing reaction and to expand the D-loop.The D-loop intermediate can be resolved by two mechanistically distinct pathways. In the synthesis-dependent singlestrand annealing pathway, the invading DNA strand with its newly synthesized DNA extension is displaced from the information donor and subsequently annealed to the ssDNA tail that adjoins the second end of the break. The synthesis-dependent single-strand annealing mode of HR does not have the potential of generating crossover recombinants. In the DNA doublestrand break repair pathway, the D-loop is maintained and recruits the second ssDNA tail by annealing, to assemble a double Holliday junction. The resolution of the double Holliday junction by a resolvase then yields a mixture of crossover and noncrossover recombinants (1).The Rad52 protein of Saccharomyces cerevisiae is needed for two di...

Pro-recombination Role of Srs2 Protein Requires SUMO (Small Ubiquitin-like Modifier) but Is Independent of PCNA (Proliferating Cell Nuclear Antigen) Interaction

“…Expression and Purification of Recombinant Proteins-The following proteins were expressed and purified as described: Srs2 and Srs2-3KR (63), Aos1/Uba2, His-Ubc9, His-FLAGSmt3 (64), His-Siz1(1-465) (55), Mre11 (56), GST-Rad52 (57), and Rad59 (65).…”

Rad52/Rad59-dependent Recombination as a Means to Rectify Faulty Okazaki Fragment Processing

“…To this end, we decided to construct an expression vector that expresses the Rad52-R70A mutant protein, which has a specific defect, i.e. impairment in annealing RPA-coated ssDNA, but is normal in recombination mediator and DNA binding activities (51). When the Rad52-R70A mutant protein was overexpressed in dna2-K1080E, it suppressed the lethality of dna2-K1080E poorly, as shown in Fig.…”

“…We were interested in the influence of Rad52 on RPAgoverned events such as DNA annealing. It was shown that RPA interferes with spontaneous annealing between two complementary ssDNA, which can be overcome by the addition of wild type Rad52 (44,51). To this end, we decided to construct an expression vector that expresses the Rad52-R70A mutant protein, which has a specific defect, i.e.…”

“…Located in the central part of Rad52, the RPA-interacting domain is responsible for the displacement of RPA from ssDNA (49,50). The RPA displacement function of Rad52 is important for both HR mediator (50) and ssDNA annealing activities (51) because the RPA-coated ssDNA is inhibitory to Rad51-ssDNA filament formation or DNA annealing between two complementary ssDNA.…”

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

“…In canonical DSB repair, Rad52 has dual functions. In addition to its role as a recombination mediator for Rad51 recruit-ment, Rad52 also participates in ssDNA annealing, an activity critical for the capture of the second DNA end at the recombination site that generates Holliday junctions (16,17). Does HR, an almost universal DNA repair mechanism in all domains of life, also occur in mitochondria?…”