Heteroduplex Joint Formation by a Stoichiometric Complex of Rad51 and Rad52 of Saccharomyces cerevisiae

Abstract

Both Rad51 and Rad52 are required for homologous genetic recombination in Saccharomyces cerevisiae. Rad51 promotes heteroduplex joint formation, a general step in homologous recombination. Rad52 facilitates the binding of Rad51 to replication protein A (RPA)-coated single-stranded DNA. The requirement of RPA can be avoided in vitro, if the single-stranded DNA is short. Using short single-stranded DNA and homologous double-stranded DNA, in the absence of RPA, we found that Rad52 (optimal at three per Rad51) was still required for Rad51-promoted heteroduplex joint formation in vitro, as assayed by the formation of D-loops, suggesting another role for Rad52. Rad51 has to bind to the single-stranded DNA before the addition of double-stranded DNA for efficient D-loop formation. Immunoprecipitation and single-stranded DNA-bead precipitation analyses revealed the presence of the free and DNA-bound complexes of Rad51 and Rad52 at a 1 to 2 stoichiometry. In the presence of single-stranded DNA, in addition to Rad51, Rad52 was required for extensive untwisting that is an intermediate step toward D-loop formation. Thus, these results suggest that the formation of the stoichiometric complex of Rad52 with Rad51 on single-stranded DNA is required for the functional binding of the protein-single-stranded DNA complex to the doublestranded DNA to form D-loops.Homologous genetic (or DNA) recombination is required for the precise repair of DNA double strand breaks and for the disjunction of homologous chromosomes during meiosis that generates genetic variation within species. A heteroduplex joint, formed by the pairing of a single-stranded DNA-tail derived from a double strand break with the complementary sequence within intact homologous double-stranded DNA, is a general intermediate of homologous recombination.In vitro, RecA of Escherichia coli efficiently promotes the formation of heteroduplex joints ("homologous pairing") between closed circular double-stranded DNA and homologous single-stranded DNA fragments (the products are called "D-loops" (1, 2)), between circular single-stranded DNA and a terminus of a homologous linear doublestranded DNA (3, 4) and in general between homologous combinations of any of single-and double-stranded DNA, independent of the strand termini (5).Saccharomyces cerevisiae Rad51, a protein homologous to RecA (6, 7), is required for meiotic recombination and recombinational repair in vivo, and in vitro, it promotes heteroduplex joint formation between circular single-stranded DNA and a terminus of linear double-stranded DNA in the presence of replication protein A (RPA, 2 a single-stranded DNA-binding protein). In this reaction, RPA is required for removing secondary structures in the circular single-stranded DNA substrate (8 -10). Rad51 alone promotes the formation of D-loops between closed circular double-stranded DNA and short single-stranded DNA fragments, although it is much less efficient than RecA (11, 12). The heteroduplex joint formation promoted by Rad51 is extensively stimulated in the pres...

Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

“…Unlike RecA, Rad51 requires Ca 2+ to enhance DNA ligation (Figure 6), and ATP suppresses overall DNA ligation (Figure 8). As for its recombinase function, Rad51 required Ca 2+ for the homologous joint formation, but this was completely substituted by the addition of another supporting protein (33,48), suggesting that Rad51 needs assistance from other proteins to perform functions fully equivalent to those of RecA. This would also be true for the enhancement of DNA ligation.…”

“…The preparation of closed circular dsDNAs from which linear dsDNA was prepared, and that of Escherichia coli RecA were cited or described previously (31). S. cerevisiae Rad51 was purified as described (33). DNA size and amount markers are Smart Ladder purchased from Nippon Gene containing linear dsDNAs of 10 000 bp (100 ng/5 μl), 8000 bp (80 ng/5 μl), 6000 bp (60 ng/5 μl), 5000 bp (50 ng/5 μl), 4000 bp (40 ng/5 μl), 3000 bp (30 ng/5 μl), 2500 bp (25 ng/5 μl), 2000 bp (20 ng/5 μl), 1500 bp (15 ng/5 μl), 1000 bp (100 ng/5 μl), 800 bp (80 ng/5 μl), 600 bp (60 ng/5 μl), 400 bp (40 ng/5 μl), 200 bp (20 ng/5 μl)).…”

“…The purification of rad51-G103E was performed as previously described for the wild-type Rad51 (33), with some modifications. We monitored rad51-G103E by measuring the ssDNA-dependent ATPase activity, and by SDS-polyacrylamide gel electrophoresis (CBB staining and immunostaining with an anti-Rad51 antibody) during the purification.…”

“…Since the binding to ssDNA and the unfolding of its secondary structures by filament formation precedes the binding of Rad51 or RecA to dsDNA (26–29), homologous joint formation is most effective when RecA or Rad51 is preincubated with ssDNA in the presence of adenosine triphosphate (ATP), and subsequently interacts with dsDNA (30). Notably, when RecA or Rad51 is first incubated with dsDNA in the presence of ATP, before the addition of ssDNA, homologous joint formation is suppressed (31–33). These similarities between RecA and Rad51 suggest that they also share a common mechanism to catalyze homologous joint formation.…”

Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein

“…The full-length S. cerevisiae Rad52, Rad51 and RPA proteins used in all in vitro experiments were expressed and purified as described previously (23,24). Rad52 233 −504 , Rad52 233– 504 F349A and Rad52 233– 504 Y409A were expressed as hexahistidine-tagged proteins, using a pET15b derivative (pET15b-PS), in which the thrombin protease recognition site was replaced with a PreScission protease recognition site by site-directed mutagenesis.…”