A RecA Homologue in Ustilago maydis That Is Distinct and Evolutionarily Distant from Rad51 Actively Promotes DNA Pairing Reactions in the Absence of Auxiliary Factors

Abstract

Two RecA homologues have been identified to date in Ustilago maydis. One is orthologous to Rad51 while the other, Rec2, is structurally quite divergent and evolutionarily distant. DNA repair and recombination proficiency in U. maydis requires both Rec2 and Rad51. Here we have examined biochemical activities of Rec2 protein purified after overexpression of the cloned gene. Rec2 requires DNA as a cofactor to hydrolyze ATP and depends on ATP to promote homologous pairing and DNA strand exchange. ATPgammaS was found to substitute for ATP in all pairing reactions examined. With superhelical DNA and a homologous single-stranded oligonucleotide as substrates, Rec2 actively promoted formation and dissociation of D-loops. When an RNA oligonucleotide was substituted it was found that R-loops could also be formed and utilized as primer/template for limited DNA synthesis. In DNA strand exchange reactions using oligonucleotides, we found that Rec2 exhibited a pairing bias that is opposite that of RecA. Single-stranded oligonucleotides were activated for DNA strand exchange when attached as tails protruding from a duplex sequence due to enhanced binding of Rec2. The results indicate that Rec2 is competent, and in certain ways even better than Rad51, in the ability to provide the fundamental DNA pairing activity necessary for recombinational repair. We propose that the emerging paradigm for homologous recombination featuring Rad51 as the essential catalytic component for strand exchange may not be universal in eukaryotes.

Role ofRAD52Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

“…Furthermore, the Rad51-K191R mutant protein catalyzes extensive strand exchange when present at high concentration. The polarity of strand exchange is 5Ј to 3Ј with respect to the complementary strand of the DNA duplex, opposite to the polarity observed for RecA but the same as for the U. maydis Rec2 protein (27,181,374). The strand exchange activity of Rad51 is greatly stimulated by the addition of RPA to the reaction mixture if RPA is added after Rad51 has already nucleated onto the initiating ssDNA substrate (373).…”

“…A proteolytic form of Rec2 purified by conventional methods from U. maydis extracts and referred to as the rec1 protein was shown to promote homologous pairing and strand exchange in vitro (180). The fulllength product of the recombinant REC2 gene catalyzes similar reactions in vitro and has the same polarity and preference for tailed duplex molecules as shown for Rad51 (27,232). The pairing and strand exchange activities of Rec2 require ATP but are not dependent on ATP hydrolysis.…”

The homologous recombination system of Ustilago maydis

“…In U. maydis Rec2 appears to be the only Rad51 paralog present in the genome (Rubin et al, 1994). Rec2 has a DNA-dependent ATPase activity, but unlike the Rad51 paralogs of yeast and human, it has an inherent ATP-dependent homologous pairing activity (Bennett and Holloman, 2001). The radiation sensitivity of rec2 can be suppressed by overexpressing Brh2, suggesting that by making more Rad51 available DNA repair can be executed in the absence of Rec2 (Kojic et al, 2006).…”

“…In early studies during the time of development of systems for exploring the biochemistry of recombination in eukaryotes, a protein preparation, which subsequently was shown to contain a proteolytic form of Rec2, was purified by conventional methods from extracts of U. maydis and was shown to promote homologous pairing and strand invasion in vitro (Bennett and Holloman, 2001).…”

Rec2 Interplay with both Brh2 and Rad51 Balances Recombinational Repair in Ustilago maydis

“…Point mutations F294A and T296A were introduced into the BRC peptide by site-directed mutagenesis using oligonucleotides and standard procedures. For expression of MBP-Rec2, the fusion utilized the complete open reading frame Rec2 1-781 derived from pCM538 (4). The open reading frame for Rec2 was introduced into pET28a (Novagen) for expression of the protein with an N-terminal His tag.…”