Homologous recombination (HR) represents an important means for the error-free elimination of DNA double-strand breaks (DSBs) and other deleterious DNA lesions from chromosomes. The Rad51 recombinase, a member of the RAD52 group of recombination proteins, catalyzes the HR reaction in the context of a helical protein polymer assembled on ssDNA that is derived from the nucleolytic processing of a primary lesion. The assembly of the Rad51-ssDNA nucleoprotein filament, often referred to as the presynaptic filament, is prone to interference by the single-strand DNA binding factor RPA. The S. cerevisiae Rad52 protein facilitates presynaptic filament assembly by helping mediate the displacement of RPA from ssDNA. On the other hand, disruption of the presynaptic filament by the Srs2 helicase leads to a net exchange of Rad51 for RPA. To understand the significance of protein-protein interactions in the control of Rad52- or Srs2-mediated presynaptic filament assembly or disassembly, we have examined two rad51 mutants, rad51 Y388H and rad51 G393D, that are simultaneously ablated for Rad52 and Srs2 interactions and one, rad51 A320V, that is differentially inactivated for Rad52 binding for their biochemical properties, and also for functional interactions with Rad52 or Srs2. We show that these mutant rad51 proteins are impervious to the mediator activity of Rad52 or the disruptive function of Srs2 in concordance with their protein interaction defects. Our results thus provide insights into the functional significance of the Rad51-Rad52 and Rad51-Srs2 complexes in the control of presynaptic filament assembly and disassembly. Moreover, our biochemical studies have helped identify A320V as a separation-of-function mutation in Rad51 with regards to a differential ablation of Rad52 interaction.
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