Sgs1 Binding to Rad51 Stimulates Homology-Directed DNA Repair in Saccharomyces cerevisiae.

Lillian Campos-Doerfler,Kristina H Schmidt,Salahuddin Syed

doi:10.1534/genetics.117.300545

Lillian Campos-Doerfler, Kristina H Schmidt + Show 1 more

Open Access

https://doi.org/10.1534/genetics.117.300545

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Journal: Genetics	Publication Date: Jan 1, 2018
Citations: 13	License type: cc-by

Affiliation: University of South Florida, Moffitt Cancer Center

Abstract

Accurate repair of DNA breaks is essential to maintain genome integrity and cellular fitness. Sgs1, the sole member of the RecQ family of DNA helicases in Saccharomyces cerevisiae, is important for both early and late stages of homology-dependent repair. Its large number of physical and genetic interactions with DNA recombination, repair, and replication factors has established Sgs1 as a key player in the maintenance of genome integrity. To determine the significance of Sgs1 binding to the strand-exchange factor Rad51, we have identified a single amino acid change at the C-terminal of the helicase core of Sgs1 that disrupts Rad51 binding. In contrast to an SGS1 deletion or a helicase-defective sgs1 allele, this new separation-of-function allele, sgs1-FD, does not cause DNA damage hypersensitivity or genome instability, but exhibits negative and positive genetic interactions with sae2Δ, mre11Δ, exo1Δ, srs2Δ, rrm3Δ, and pol32Δ that are distinct from those of known sgs1 mutants. Our findings suggest that the Sgs1-Rad51 interaction stimulates homologous recombination (HR). However, unlike sgs1 mutations, which impair the resection of DNA double-strand ends, negative genetic interactions of the sgs1-FD allele are not suppressed by YKU70 deletion. We propose that the Sgs1-Rad51 interaction stimulates HR by facilitating the formation of the presynaptic Rad51 filament, possibly by Sgs1 competing with single-stranded DNA for replication protein A binding during resection.

Highlights

Accurate repair of DNA breaks is essential to maintain genome integrity and cellular fitness
Rad51 binds to the loop that connects the helicase core of Sgs1 to the helicase- and RNaseD C-terminal (HRDC) domain
We have identified a novel separation-of-function mutant of Sgs1 that fails to interact with Rad51, but does not cause the severe sensitivity to DNA-damaging agents seen in cells lacking Sgs1 or expressing helicase-defective Sgs1

Summary

Introduction

Accurate repair of DNA breaks is essential to maintain genome integrity and cellular fitness. DNA double-strand breaks (DSBs) can be induced exogenously by DNA-damaging agents, or form endogenously if the replisome collapses at a nick in the template strand or encounters a physical barrier that blocks progression of the replisome, such as a bound protein, DNA adduct, interstrand cross-link, or unusual DNA structure Cells can repair such DSBs by homologous recombination (HR) or nonhomologous endjoining (NHEJ). In S phase, Ku and MRX bind to the DSB first and recruitment of Sae activates short-range resection, removing Ku and MRX, and leaving a small 39 single-stranded DNA (ssDNA) overhang (Trujillo et al 2003; Mimitou and Symington 2008, 2010; Nicolette et al 2010; Niu et al 2010; Cannavo and Cejka 2014). Top topoisomerase, and the nucleotide excision repair factor Rad have been shown to physically interact with the N-terminal tail of Sgs, whereas Rad and Mlh binding has been narrowed down to the region C-terminal to the helicase core (Watt et al 1995; Fricke et al 2001; Saffi et al 2001; Wu et al 2001; Dherin et al 2009; Hegnauer et al 2012)

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