Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption.
The SRS2 (Suppressor of RAD Six screen mutant 2) gene encodes an ATP-dependent DNA helicase that regulates homologous recombination in Saccharomyces cerevisiae. Mutations in SRS2 result in a hyper-recombination phenotype, sensitivity to DNA damaging agents and synthetic lethality with mutations that affect DNA metabolism. Several of these phenotypes can be suppressed by inactivating genes of the RAD52 epistasis group that promote homologous recombination, implicating inappropriate recombination as the underlying cause of the mutant phenotype. Consistent with the genetic data, purified Srs2 strongly inhibits Rad51-mediated recombination reactions by disrupting the Rad51-ssDNA presynaptic filament. Srs2 interacts with Rad51 in the yeast two-hybrid assay and also in vitro. To investigate the functional relevance of the Srs2-Rad51 complex, we have generated srs2 truncation mutants that retain full ATPase and helicase activities, but differ in their ability to interact with Rad51. Importantly, the srs2 mutant proteins attenuated for Rad51 interaction are much less capable of Rad51 presynaptic filament disruption. An internal deletion in Srs2 likewise diminishes Rad51 interaction and anti-recombinase activity. We also present evidence that deleting the Srs2 C-terminus engenders a hyper-recombination phenotype. These results highlight the importance of Rad51 interaction in the anti-recombinase function of Srs2, and provide evidence that this Srs2 function can be uncoupled from its helicase activity.
Colavito
Sierra
S
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA.
Macris-Kiss
Margaret
M
Seong
Changhyun
C
Gleeson
Olive
O
Greene
Eric C
EC
Klein
Hannah L
HL
Krejci
Lumir
L
Sung
Patrick
P
D000251:Adenosine Triphosphatases / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000378:metabolism* / Q000648:ultrastructure; D045522:Recombinases / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques
D045522:Recombinases; D029701:Saccharomyces cerevisiae Proteins; C061376:SRS2 protein, S cerevisiae; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases; D004265:DNA Helicases