Dynamic Regulatory Interactions of Rad51, Rad52, and Replication Protein-A in Recombination Intermediates

Abstract

Rad51, Rad52, and replication protein-A (RPA) play crucial roles in the repair of DNA double-strand breaks in Saccharomyces cerevisiae. Rad51 mediates DNA strand exchange, a key reaction in DNA recombination. Rad52 recruits Rad51 into single-stranded DNAs (ssDNAs) that are saturated with RPA. Rad52 also promotes annealing of ssDNA strands that are complexed with RPA. Specific protein–protein interactions are involved in these reactions. Here we report new biochemical characteristics of these protein interactions. First, Rad52–RPA interaction requires multiple molecules of RPA to be associated with ssDNA, suggesting that multiple contacts between the Rad52 ring and RPA–ssDNA filament are needed for stable binding. Second, RPA-t11, which is a recombination-deficient mutant of RPA, displays a defect in interacting with Rad52 in the presence of salt above 50 mM, explaining the defect in Rad52-mediated ssDNA annealing in the presence of this mutation. Third, ssDNA annealing promoted by Rad52 is preceded by aggregation of multiple RPA–ssDNA complexes with Rad52, and Rad51 inhibits this aggregation. These results suggest a regulatory role for Rad51 that suppresses ssDNA annealing and facilitates DNA strand invasion. Finally, the Rad51–double-stranded DNA complex disrupts Rad52–RPA interaction in ssDNA and titrates Rad52 from RPA. This suggests an additional regulatory role for Rad51 following DNA strand invasion, where Rad51–double-stranded DNA may inhibit illegitimate second-end capture to ensure the error-free repair of a DNA double-strand break.