Cooperative interactions facilitate stimulation of Rad51 by the Swi5-Sfr1 auxiliary factor complex

Abstract

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. We present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a conserved auxiliary factor. Two distinct sites within the intrinsically disordered N-terminus of Sfr1 (Sfr1N) were found to cooperatively bind Rad51. Deletion of this domain impaired Rad51 stimulation in vitro and rendered cells sensitive to DNA damage. By contrast, amino acid-substitution mutants, which had comparable biochemical defects, could promote DNA repair, suggesting that Sfr1N has another role in addition to Rad51 binding. Unexpectedly, the DNA repair observed in these mutants was dependent on Rad55-Rad57, another auxiliary factor complex hitherto thought to function independently of Swi5-Sfr1. When combined with the finding that they form a higher-order complex, our results imply that Swi5-Sfr1 and Rad55-Rad57 can collaboratively stimulate Rad51 in Schizosaccharomyces pombe.

A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors

“…Because some auxiliary factors that contain the FxxA motif employ Rad51 mimicry to interact with Rad51, FxxA has been proposed to function as a Rad51 interaction motif ( Pellegrini et al, 2002 ; Shin et al, 2003 ). Neither Swi5 nor Sfr1 contain the FxxA consensus sequence and we recently identified two non-FxxA sites within the intrinsically disordered N-terminal half of Sfr1 that are responsible for the binding of Swi5-Sfr1 to Rad51 ( Argunhan et al, 2020 ). By contrast, both Rad57 ( 145 FELA 148 ) and Rad52 ( 17 FNTA 20 and 338 FISA 341 ) contain the FxxA consensus sequence, as does Rad54 ( 804 FIRA 807 ).…”

“…Notably, the PAP is important for interactions with auxiliary factors involved in both the early (Rad52 and Rad55-Rad57) and late (Rad54) stages of DNA strand exchange, indicating that the PAP is integral to the role of Rad51 in HR. Further research is needed to examine the temporospatial coordination of Rad51 binding, especially in light of the finding that some auxiliary factors such as Rad55-Rad57 and Swi5-Sfr1 interact with each other ( Argunhan et al, 2020 ).…”

“…Suppression of the DNA damage sensitivity associated with rad51-E206A by Sfr1 contradicts the commonly accepted model wherein Rad55-Rad57 and Swi5-Sfr1 comprise independent sub-pathways of HR ( Akamatsu et al, 2003 ; Akamatsu et al, 2007 ). We recently showed that Rad55-Rad57 can suppress defects in the interaction between Swi5-Sfr1 and Rad51, and that Swi5-Sfr1 physically interacts with Rad55-Rad57 ( Argunhan et al, 2020 ). Thus, there is increasing evidence to suggest that Rad55-Rad57 and Swi5-Sfr1, while capable of functioning independently of each other, collaboratively promote Rad51-dependent DNA repair.…”

“…Each aliquot was then resuspended in 35 mL fresh YES and allowed to recover at 30°C for 3 hr with shaking. Cells were then harvested and treated as previously described ( Argunhan et al, 2020 ). Briefly, 1 × 10 8 cells were resuspended in 1 mL of ice-cold water and mixed with 150 µL of 1.85 M NaOH 7.5% β-mercaptoethanol on ice for 15 min.…”

“…By contrast, the rad57Δ and sfr1Δ mutants show only moderate sensitivity to DNA damage, while the rad57Δ sfr1Δ double mutant is as sensitive as the rad51Δ single mutant. Based on this additivity, it was proposed that Rad55-Rad57 and Swi5-Sfr1 comprise independent sub-pathways of HR that function in parallel to promote Rad51-dependent DNA repair ( Akamatsu et al, 2003 ; Akamatsu et al, 2007 ), although recent evidence has evoked a re-examination of this model ( Argunhan et al, 2020 ).…”