The Rad51 paralogs facilitate a novel DNA strand specific damage tolerance pathway

Joel C Rosenbaum,Steven A Roberts,Bennett Van Houten,Sauvik Das ,Daniel P Normolle,Alessio De Magis,Patrick Sung,Timothy C Ratterman,Tony M Mertz,Katrin Paeschke,Ellen Macnary,Catherine A Pressimone,Youngho Kwon,Andrew P Vandemark,Hinke G Kazemier,Sarah R Hengel ,Braulio Bonilla,Benjamin W Herken,Stephen K Godin,Kara A Bernstein

doi:10.1038/s41467-019-11374-8

Abstract

Accurate DNA replication is essential for genomic stability and cancer prevention. Homologous recombination is important for high-fidelity DNA damage tolerance during replication. How the homologous recombination machinery is recruited to replication intermediates is unknown. Here, we provide evidence that a Rad51 paralog-containing complex, the budding yeast Shu complex, directly recognizes and enables tolerance of predominantly lagging strand abasic sites. We show that the Shu complex becomes chromatin associated when cells accumulate abasic sites during S phase. We also demonstrate that purified recombinant Shu complex recognizes an abasic analog on a double-flap substrate, which prevents AP endonuclease activity and endonuclease-induced double-strand break formation. Shu complex DNA binding mutants are sensitive to methyl methanesulfonate, are not chromatin enriched, and exhibit increased mutation rates. We propose a role for the Shu complex in recognizing abasic sites at replication intermediates, where it recruits the homologous recombination machinery to mediate strand specific damage tolerance.

Highlights

Accurate DNA replication is essential for genomic stability and cancer prevention
To determine the role of DNA binding in S. cerevisiae Shu complex function, we modeled the putative DNA-binding loops of the Shu complex members, the Rad[51] paralogs, Csm[2] and Psy[3]
Since we find that the Shu complex binds most tightly to double-flap DNA12,19 and is important for resistance to abasic sites (Fig. 2a), we hypothesized that Csm2-Psy[3] may be enriched at chromatin when abasic sites accumulate during replication

Summary

Introduction

Accurate DNA replication is essential for genomic stability and cancer prevention. Homologous recombination is important for high-fidelity DNA damage tolerance during replication. We provide evidence that a Rad[51] paralog-containing complex, the budding yeast Shu complex, directly recognizes and enables tolerance of predominantly lagging strand abasic sites. We propose a role for the Shu complex in recognizing abasic sites at replication intermediates, where it recruits the homologous recombination machinery to mediate strand specific damage tolerance. We provide evidence that DNA-binding components of the budding yeast Shu complex, Csm2-Psy[3], bind double-flap DNA substrates containing an abasic site analog, and increase chromatin association when abasic sites accumulate. We show that Csm2-Psy[3] aids in preventing TLSinduced mutations that arise in the lagging strand during replication of DNA templates containing abasic sites. We propose a model whereby the Shu complex directly recognizes abasic sites on the lagging strand of a replication fork to facilitate a error-free, strand-specific damage tolerance pathway

Methods

Results

Conclusion