Rad51 Protein from the Thermotolerant Yeast Pichia angusta as a Typical but Thermodependent Member of the Rad51 Family

Abstract

ABSTRACT The Rad51 protein from the methylotrophic yeast Pichia angusta (Rad51 Pa ) of the taxonomic complex Hansenula polymorpha is a homolog of the RecA-RadA-Rad51 protein superfamily, which promotes homologous recombination and recombination repair in prokaryotes and eukaryotes. We cloned the RAD51 gene from the cDNA library of the thermotolerant P. angusta strain BKM Y1397. Induction of this gene in a rad51 -deficient Saccharomyces cerevisiae strain partially complemented the survival rate after ionizing radiation. Purified Rad51 Pa protein exhibited properties typical of the superfamily, including the stoichiometry of binding to single-stranded DNA (ssDNA) (one protomer of Rad51 Pa per 3 nucleotides) and DNA specificity for ssDNA-dependent ATP hydrolysis [poly(dC) > poly(dT) > φX174 ssDNA > poly(dA) > double-stranded M13 DNA]. An inefficient ATPase and very low cooperativity for ATP interaction position Rad51 Pa closer to Rad51 than to RecA. Judging by thermoinactivation, Rad51 Pa alone was 20-fold more thermostable at 37°C than its S. cerevisiae homolog (Rad51 Sc ). Moreover, it maintained ssDNA-dependent ATPase and DNA transferase activities up to 52 to 54°C, whereas Rad51 Sc was completely inactive at 47°C. A quick nucleation and an efficient final-product formation in the strand exchange reaction promoted by Rad51 Pa occurred only at temperatures above 42°C. These reaction characteristics suggest that Rad51 Pa is dependent on high temperatures for activity.