Rad54 and Rdh54 prevent Srs2-mediated disruption of Rad51 presynaptic filaments

Abstract

Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombination intermediates. Rad54 and Rdh54 bind tightly to Rad51-ssDNA and act downstream of Srs2, suggesting that they may affect the ability of Srs2 to dismantle Rad51 filaments. Here, we used DNA curtains to determine whether Rad54 and Rdh54 alter the ability of Srs2 to disrupt Rad51 filaments. We show that Rad54 and Rdh54 act synergistically to greatly restrict the antirecombinase activity of Srs2. Our findings suggest that Srs2 may be accorded only a limited time window to act and that Rad54 and Rdh54 fulfill a role of prorecombinogenic licensing factors.

Rdh54 stabilizes Rad51 at displacement loop intermediates to regulate genetic exchange between chromosomes

“…Physical conflicts between the myriad of proteins present on DNA limit its accessibility and can impede the successful repair of damage during HR. Specific examples of this include the inhibition of Sgs1 translocation by Rad52 [52], the inhibition of Srs2 translocation by Rad54 and Rdh54 [42], and the inhibition of both Srs2 and Sgs1 by the meiosis specific recombinase Dmc1 [53]. Our data indicates that Rdh54 acts as a barrier to Rad54 at newly formed D-loop intermediates, consistent with previous studies that showed Rdh54 limited D-loop size [54].…”

“…Rad54 is thought to remove Rad51 by induced ATP hydrolysis of Rad51 in a mechanism resembling the activity of RecA [5, 15, 55, 56]. In contrast, Rdh54 has previously been reported to stabilize Rad51-ssDNA filaments [42], although the exact mechanism is unclear. Several possibilities include the stabilization of Rad51 against ATP hydrolysis [42], the physical interference of Rad54 translocation, or by limiting Rad54 movement through the interaction between Rdh54 and Rad51.…”

“…In contrast, Rdh54 has previously been reported to stabilize Rad51-ssDNA filaments [42], although the exact mechanism is unclear. Several possibilities include the stabilization of Rad51 against ATP hydrolysis [42], the physical interference of Rad54 translocation, or by limiting Rad54 movement through the interaction between Rdh54 and Rad51. Although somewhat controversial, a link between Rdh54 and locating donor DNA has also been reported using a donor less strain of yeast [57, 58], suggesting stabilization of Rad51 filaments could have a limited role in the process of locating the correct donor DNA molecule.…”

“…Several biochemical differences have also been observed between Rdh54 and Rad54. These include the observation that Rad54 preferentially initiates Rad51 mediated homology search [4, 41], that Rdh54 stabilizes Rad51 filaments on ssDNA against ATP depletion, and that each translocase shares a unique binding site on Rad51 filaments [42]. Major differences in RDH54 and RAD54 activity manifests in cells during HR.…”

Rad51 and Dmc1 have similar tolerance for mismatches in yeast meiosis

“…A set of mediator proteins (in budding yeast, Rad52, Rad55 and Rad57) enable Rad51 to assemble into a nucleoprotein filament on the single-stranded ends of a DSB that are created by 5' to 3' resection (15,(22)(23)(24). Rad51's ability to promote DNA strand exchange also depends on the Rad54 chromatin remodeler, which binds to Rad51 and promotes strand invasion into homologous sequences and later branch migration steps in recombination (25)(26)(27)(28)(29). In contrast, meiotic recombination in many eukaryotes, including both budding yeast and mammals, recombination depends on Dmc1, a Rad51 paralog (19,30).…”