Rad52-Rad51 association is essential to protect Rad51 filaments against Srs2, but facultative for filament formation

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.

Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

“…We have previously shown that Srs2 removes Rad51 I345T from ssDNA at a reduced velocity (∼85 nt·s −1 ) compared with the velocity observed with WT Rad51 (∼140 nt·s −1 ) (44), indicating that Rad51 I345T slows but does not stop the movement of Srs2, which is consistent with the observed reduction in Srs2mediated ATP hydrolysis in the presence of Rad51 I345T . Together, these findings suggest that Rad55/57 (22), Rad52 (24,42), and Rad51 I345T (present study and ref. 44) do not function by inhibiting Srs2 ATP hydrolysis activity.…”

“…Rad55/57 and Rad52 have been implicated as potential regulators of Srs2 activity, albeit through poorly understood mechanisms (21,22). Interestingly, Rad55/57 and Rad52 have no impact on Srs2 ATP hydrolysis rates (22,24,42), and Srs2 readily removes Rad52 from RPA-ssDNA complexes (42). Rad51 I345T is a suppressor mutation that bypasses the need for Rad55/57 (22,43).…”

“…Mechanistically, Dmc1 acts by inhibiting Srs2 ATP hydrolysis, which in turn prevents Srs2 from translocating on ssDNA bound by Dmc1. The ability to shut down Srs2 translocation through inhibition of its ATP hydrolysis activity appears to distinguish Dmc1 from other known negative regulators of Srs2 antirecombinase activities, such as Rad55/5 or Rad52, which do not appear to affect the ssDNAdependent ATP hydrolysis activity of Srs2 (22,24,42). Our findings suggest that this inhibition likely takes place as a result of direct physical contact between the advancing Srs2 motor proteins and the 3′ ends of Dmc1 filaments, suggesting a mechanism involving allosteric communication that turns off ATP hydrolysis by Srs2 (Fig.…”

“…Srs2 antirecombinase activity is counterbalanced by the Rad51 paralog complex Rad55/57, the Shu complex (composed of the Rad51 paralogs Psy3 and Csm2 and the SWIM-domain proteins Shu1 and Shu2), and Rad52, all of which enhance assembly or stability of early HR intermediates ( Fig. 1A) (21)(22)(23)(24). The role of Srs2 in helping to minimize cross-overs during mitosis is well established, but its functions during meiosis remain poorly understood (15).…”

The PCNA unloader Elg1 promotes recombination at collapsed replication forks in fission yeast

“…Rad52 aids this process by mediating the loading of Rad51 onto ssDNA coated with the ssDNA binding protein RPA (Krogh and Symington, 2004). It also helps protect Rad51-ssDNA filaments from disruption by the anti-recombinogenic DNA helicases Srs2 and Fbh1 (Lorenz et al, 2009; Ma et al, 2018; Osman et al, 2005).…”