Physical interaction between replication protein A and Rad51 promotes exchange on single-stranded DNA.

Stauffer ME, Chazin WJ
J Biol Chem. 2004 279 (24): 25638-45

PMID: 15056657 · DOI:10.1074/jbc.M400029200

Replication protein A (RPA) is displaced from single-stranded DNA (ssDNA) by Rad51 during the initiation of homologous recombination. Interactions between these proteins have been reported, but the functional significance of the direct RPA-Rad51 interaction has yet to be elucidated. We have identified and characterized the interaction between DNA-binding domain A of RPA (RPA70A) and the N-terminal domain of Rad51 (Rad51N). NMR chemical shift mapping showed that Rad51N binds to the ssDNA-binding site of RPA70A, suggesting a competitive mechanism for the displacement of RPA from ssDNA by Rad51. A structure of the RPA70A-Rad51N complex was generated by experimentally guided modeling and then used to design mutations that disrupt the binding interface. Functional ATP hydrolysis assays were performed for wild-type Rad51 and a mutant defective in binding RPA. Rates of RPA displacement for the mutant were significantly below those of wild-type Rad51, suggesting that a direct RPA-Rad51 interaction is involved in displacing RPA in the initiation stage of genetic recombination.

MeSH Terms (10)

Adenosine Triphosphate Binding, Competitive Binding Sites DNA, Single-Stranded DNA-Binding Proteins Magnetic Resonance Spectroscopy Point Mutation Rad51 Recombinase Recombination, Genetic Replication Protein A

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