Structure/function studies on the allosteric mechanism of Rad51 recombinase (549.8)

Abstract

Eukaryotic Rad51 recombinase plays a central role in maintaining genome stability by promoting homology‐directed repair of DNA double‐strand breaks and stalled replication forks. Rad51 forms presynaptic filaments on single‐stranded DNA, which activates the ATPase, homologous pairing, and DNA strand exchange activities of the enzyme. Structure/function studies of yeast Rad51 reveal that coordination between the ATP and DNA binding sites is mediated by residues at the protomer‐protomer interface and in DNA binding loops L1 and L2. Conserved interface residues Phe‐187 and His‐352 play central roles in sensing the ATP‐bound state of Rad51‐ssDNA filaments and inducing conformational changes in the filament that are necessary for DNA strand exchange. An F187A mutant is proficient in ssDNA‐dependent ATPase activity but deficient in DNA strand exchange, suggesting an inability to couple ATP binding to the homology search function of Rad51. Mutations at the His‐352 locus affect ssDNA‐binding affinity and catalytic turnover. An H352Y mutant catalyzes a single round of DNA strand exchange and subsequently fails due to a nucleotide exchange defect. Meanwhile, new X‐ray crystallographic structures reveal previously unseen details of the DNA and ATP binding surfaces of Rad51. The structures indicate that conserved aromatic residue Phe‐290 is important for stabilizing a conformation of L1 that allows optimal contacts between conserved Arg‐293 and DNA. Results of this work will facilitate modeling of the structural transitions that occur in Rad51 filaments during recombination.Grant Funding Source: NIH grant no. P02 CA098993