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Abstract
The basidiomycete Ustilago maydis contains at least two homologues of the E. coli RecA protein. While one is structurally similar to Rad51, the other, Rec2, is much more structurally divergent and apparently without a close relative among RecA homologues. In this study, the biochemical nature of the Rec2 protein is examined, after overexpression of the cloned gene, to further define its role in the mechanism of homologous recombination. Recombinant Rec2 exhibited DNA-dependent ATPase activity with an estimated kcat of 1.1 min −1 in the presence of single-stranded DNA and promoted the pairing and strand exchange of homologous DNA molecules. Nucleotide hydrolysis was not essential to pairing and strand exchange since ATPγS could substitute for ATP in all reactions examined. The Rec2 protein actively promoted formation and dissociation of D-loops with superhelical DNA and a homologous single-stranded oligonucleotide. Additionally, Rec2 promoted R-loop formation between an RNA oligonucleotide and homologous superhelical DNA, and these joint molecules could be subsequently used as a substrate for DNA replication. In strand exchange reactions between oligonucleotide substrates, Rec2 exhibited an opposite polarity of pairing than that observed with RecA. Furthermore, strand exchange was enhanced by incorporation of single-stranded DNA into duplex DNA with a single-stranded tail. Addition of RPA to reactions inhibited Rec2-mediated pairing and strand exchange. These results indicate that Rec2 has the innate ability to promote the homologous recognition and the strand invasion steps envisioned as being central to the mechanism of DNA double-strand break repair by homologous recombination. Therefore, the emerging view that Rad51 is the sole catalytic component for strand exchange during mitotic homologous recombination while other RAD52 epistasis group gene products serve as accessory proteins to enhance Rad51 function may not be universal in eukaryotes.