The rad51-K191R ATPase-defective mutant is impaired for presynaptic filament formation.
The nucleoprotein filament formed by Rad51 polymerization on single-stranded DNA is essential for homologous pairing and strand exchange. ATP binding is required for Rad51 nucleoprotein filament formation and strand exchange, but ATP hydrolysis is not required for these functions in vitro. Previous studies have shown that a yeast strain expressing the rad51-K191R allele is sensitive to ionizing radiation, suggesting an important role for ATP hydrolysis in vivo. The recruitment of Rad51-K191R to double-strand breaks is defective in vivo, and this phenotype can be suppressed by elimination of the Srs2 helicase, an antagonist of Rad51 filament formation. The phenotype of the rad51-K191R strain is also suppressed by overexpression of Rad54. In vitro, the Rad51-K191R protein exhibits a slight decrease in binding to DNA, consistent with the defect in presynaptic filament formation. However, the rad51-K191R mutation is dominant in heterozygous diploids, indicating that the defect is not due simply to reduced affinity for DNA. We suggest the Rad51-K191R protein either forms an altered filament or is defective in turnover, resulting in a reduced pool of free protein available for DNA binding.
Fung
Cindy W
CW
Graduate Program in Cellular, Molecular and Biophysical Studies, Columbia University Medical Center, New York, NY 10032, USA.
Fortin
Gary S
GS
Peterson
Shaun E
SE
Symington
Lorraine S
LS
D000251:Adenosine Triphosphatases / Q000172:deficiency* / Q000235:genetics / Q000502:physiology*; D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics*; D001120:Arginine / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics; D045643:DNA Repair Enzymes; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005720:Gamma Rays; D017353:Gene Deletion; D008239:Lysine / Q000235:genetics; D009154:Mutation; D009698:Nucleoproteins / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics; D051135:Rad51 Recombinase / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D029701:Saccharomyces cerevisiae Proteins; C061376:SRS2 protein, S cerevisiae; D001120:Arginine; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes; D008239:Lysine