Abstract
Homology search and strand exchange mediated by Rad51 nucleoprotein filaments are key steps of the homologous recombination process. In budding yeast, Rad52 is the main mediator of Rad51 filament formation, thereby playing an essential role. The current model assumes that Rad51 filament formation requires the interaction between Rad52 and Rad51. However, we report here that Rad52 mutations that disrupt this interaction do not affect γ-ray- or HO endonuclease-induced gene conversion frequencies. In vivo and in vitro studies confirmed that Rad51 filaments formation is not affected by these mutations. Instead, we found that Rad52-Rad51 association makes Rad51 filaments toxic in Srs2-deficient cells after exposure to DNA damaging agents, independently of Rad52 role in Rad51 filament assembly. Importantly, we also demonstrated that Rad52 is essential for protecting Rad51 filaments against dissociation by the Srs2 DNA translocase. Our findings open new perspectives in the understanding of the role of Rad52 in eukaryotes.
Highlights
Homologous recombination (HR) is a highly conserved mechanism for the repair of DNA doublestrand breaks and stalled replication forks
Rad52-Y376A restores resistance to DNA damage in srs2D homozygous diploid cells exposed to g-rays as well as viability of srs2D haploid cells in which a HO-induced DSB is repaired by single strand annealing (SSA)
The rad52-Y376A mutation suppresses synthetic lethality induced in Srs2-deficient cells by mutations in genes involved in HR or in DNA replication
Summary
Homologous recombination (HR) is a highly conserved mechanism for the repair of DNA doublestrand breaks and stalled replication forks. The unproductive association of Rad with ssDNA could interfere with the normal progression of DNA replication forks or DNA repair (Aboussekhra et al, 1992; Klein, 2001; Vasianovich et al, 2017) This control of toxic filaments suggests that functional Rad filaments are protected from Srs activity. We proposed that this association is responsible for the toxicity of Rad filaments in Srs2-deficient cells because Rad mutations that do not affect Rad filament formation suppress the sensitivity of Srs2-deficient cells to DNA-damaging agents. We sought to better understand the role of Rad in the formation of toxic Rad filaments To this aim, we used a random mutagenesis approach to select new RAD52 gene mutations that suppress the sensitivity of Srs2-deficient cells to DNA damage. The newly identified Rad role could be extended to other eukaryotes and to other mediator proteins
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