Rad51 protein from the thermotolerant yeast Pichia angusta as a typical but thermodependent member of the Rad51 family.
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) > phiX174 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 degrees C than its S. cerevisiae homolog (Rad51(Sc)). Moreover, it maintained ssDNA-dependent ATPase and DNA transferase activities up to 52 to 54 degrees C, whereas Rad51(Sc) was completely inactive at 47 degrees 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 degrees C. These reaction characteristics suggest that Rad51(Pa) is dependent on high temperatures for activity.
Shalguev
Valery I
VI
Division of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute, Russian Academy of Sciences, Gatchina/St. Petersburg 188300, Russia.
Kil
Yury V
YV
Yurchenko
Ludmila V
LV
Namsaraev
Eugene A
EA
Lanzov
Vladislav A
VA
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D018076:DNA, Complementary / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005720:Gamma Rays; D015723:Gene Library; D005816:Genetic Complementation Test; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D051135:Rad51 Recombinase; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases