Promotion of presynaptic filament assembly by the ensemble of S. cerevisiae Rad51 paralogues with Rad52

William A Gaines,Stephen K Godin,Faiz F Kabbinavar,Kara A Bernstein,Patrick Sung,Timsi Rao,Andrew P Vandemark

doi:10.1038/ncomms8834

William A Gaines, Stephen K Godin + Show 5 more

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https://doi.org/10.1038/ncomms8834

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Journal: Nature Communications	Publication Date: Jul 28, 2015
Citations: 66	License type: CC BY 4.0

Affiliation: Yale University, University of Pittsburgh

Abstract

The conserved budding yeast Rad51 paralogues, including Rad55, Rad57, Csm2 and Psy3 are indispensable for homologous recombination (HR)-mediated chromosome damage repair. Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2. Here we show that Rad55 bridges an interaction between Csm2 with Rad51 and Rad52 and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55–Rad57 and Rad52 to promote nucleation of Rad51 on single-stranded DNA pre-occupied by replication protein A (RPA). The csm2–F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo. Our results reveal that Rad55–Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.

Highlights

The conserved budding yeast Rad[51] paralogues, including Rad[55], Rad[57], Csm[2] and Psy[3] are indispensable for homologous recombination (HR)-mediated chromosome damage repair
We first focused on the purified Csm2–Psy[3] heterodimer (Supplementary Fig. 1a), as the known protein interaction and DNA-binding activities occur through these subunits, including our previous findings that Csm[2] associates with Rad[55] in the Y2H system[16]
We have furnished evidence that while the Shu complex is devoid of recombination mediator activity, it synergizes with Rad55–Rad[57] and Rad[52] to mediate the assembly of Rad[51] on replication protein A (RPA)-coated single-stranded DNA (ssDNA) (Supplementary Fig. 8)

Summary

Introduction

The conserved budding yeast Rad[51] paralogues, including Rad[55], Rad[57], Csm[2] and Psy[3] are indispensable for homologous recombination (HR)-mediated chromosome damage repair. We show that Rad[55] bridges an interaction between Csm[2] with Rad[51] and Rad[52] and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55–Rad[57] and Rad[52] to promote nucleation of Rad[51] on singlestranded DNA pre-occupied by replication protein A (RPA). Our results reveal that Rad55–Rad[57], the Shu complex and Rad[52] act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition. Rad[51] presynaptic filament assembly on ssDNA pre-occupied by RPA, the ubiquitous singlestrand DNA-binding protein, is facilitated by Rad[52] (or by BRCA2 in humans) and a heterodimeric complex of the Rad[51] paralogues Rad[55] and Rad[57] (refs 2,8). Our data support a model wherein Csm[2] and Rad[55] bridge interactions among the Rad55–Rad[57] heterodimer, the Shu complex and Rad[52] to promote Rad51-dependent homology-directed DNA repair

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