Role of Yeast Rad51 Interface Residue F187A and DNA Binding Loop 1 Residue F290 in Coordinating DNA Binding, ATPase, and DNA Strand Exchange Activities

Abstract

The recombinase Rad51 promotes homologous recombination in eukaryotes. Recombination is initiated when Rad51 forms a presynaptic filament on ssDNA. Filament formation activates the enzymatic activities of Rad51 including ssDNA-dependent ATP hydrolysis, homology search and DNA strand exchange. Proper filament assembly is crucial for double-strand break repair and the avoidance of cancer. Previous studies showed that the yeast Rad51 H352 residue, located at the protomer-protomer interface near the ATPase site, is important for allosteric communication. The H352Y variant exhibits increased affinity for ssDNA but is unable to undergo multiple turnovers of ATP hydrolysis. The crystal structure of H352Y shows that F187, another conserved residue at the interface, may also be a significant player. In this study, we show that the F187A variant can hydrolyze ATP but has a decreased affinity for DNA and thus a deficiency in strand exchange activity is seen. Additionally, we report a novel X-ray crystal structure of yeast Rad51 resolving the DNA binding loop 1. This structure reveals a conserved aromatic residue, F290 that contacts the residues of the ATPase site. Like wild-type Rad51, the F290A variant possess ATP-dependent binding to both dsDNA and ssDNA. Unlike wild-type, however, F290A is not active for ATPase and DNA strand exchange activities. Data suggest that the deficient strand exchange activities for both variants is due to the disruption of the signaling pathway between the binding sites for ATP and DNA. Funded by NIH.