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

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 ssDNAdependent ATP hydrolysis [poly(dC) > poly(dT) > X174 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.Pichia angusta, one of the species of the taxonomic complex Hansenula polymorpha (25), is a methylotrophic yeast which has attracted considerable attention as a promising host for the production of recombinant proteins because of its powerful promoter elements, its ability to grow at high density on inexpensive substrates, and the unusual properties of its homologous and nonhomologous recombination systems promoting a multiple tandem integration of a nonlinearized plasmid in the host chromosome (for reviews, see references 11, 28, and 31). One additional characteristic that might have a biotechnological advantage is its thermotolerance, an ability to grow at temperatures (42°C and higher) which are not acceptable for other yeast strains.Among 2,500 P. angusta novel protein-encoding genes now identified, 6% have no homologs in Saccharomyces cerevisiae (3). Little is known about the recombination or DNA repair proteins in P. angusta, such as the homologous DNA transferase Rad51 and its paralogs. This recombinase is a RecARadA-like protein, found in all three domains of life (4), which forms filaments on single-stranded DNA (ssDNA) in the presence of ATP (and thus possesses ATPase activity) and promotes homologous pairing and strand exchange, which are the two main steps in the initiation of homologous recombination and recombination repair, as well (6,16,27). All members of the RecA-RadA-Rad51 recombinase superfamily form nucleoprotein filaments of similar structures and stoichiometries, and they display similar preferences in DNA substrates for DNAdependent ATP hy...

Characterization of recombinase DMC1B and its functional role as Rad51 in DNA damage repair in Giardia duodenalis trophozoites

“…This outcome was expected because Morais [45] did not obtain any complementation after expressing bacterial recA protein in a rad51 defective yeast strain. Similarly, a rad51 ortholog from Pichia angusta failed to complement a Drad51 Saccharomyces cerevisiae strain [46], possibly due to differences in sequences that could impair interactions with repair proteins from yeast, thus indicating that these interactions are species-specific. One of these interactions occurs in the NTD of RAD51 with RPA1 [42], and at least preliminarily, we found that rGdRPA1 and rGdDMC1B interact (data not shown).…”

Flip/flop mating-type switching in the methylotrophic yeast Ogataea polymorpha is regulated by an Efg1-Rme1-Ste12 pathway

“…polymorpha RAD51 gene has previously been reported to partially complement an S . cerevisiae rad51 mutant, and the protein catalyzes DNA strand exchange in vitro [ 47 ]. Rad17 is a component of the checkpoint signaling clamp called 9-1-1 in humans or Ddc1-Mec3-Rad17 in S .…”