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

Bilge Argunhan,Negar Afshar,Masayuki Takahashi,Hideo Takahashi,Hiroshi Iwasaki,Masayoshi Sakakura,Yasuto Murayama,Kentaro Ito,Shuji Kanamaru,Misato Kurihara,Takahisa Maki,Hideo Tsubouchi

doi:10.7554/elife.52566

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.

Highlights

DNA double-strand breaks (DSBs) are a toxic form of DNA damage in which a DNA molecule is broken into two fragments
After initial pairing with the complementary strand of the double-stranded DNA (dsDNA), the Rad51 filament further displaces the noncomplementary strand by driving strand transfer, resulting in the formation of an intermediate structure known as a displacement loop
Sfr1N is essential for the role of Swi5-Sfr1 in DNA repair

Summary

Introduction

DNA double-strand breaks (DSBs) are a toxic form of DNA damage in which a DNA molecule is broken into two (or more) fragments. During HR, an intact stretch of DNA that shares sequence similarity to the DSB site is identified and utilized as a template for synthesis-dependent repair. HR is initiated by the formation of 3’ single-stranded DNA (ssDNA) at the DSB site. This ssDNA is first bound by RPA, by the ubiquitous RecA-family recombinase Rad, which forms a righthanded nucleoprotein filament. The Rad filament is able to capture intact double-stranded DNA (dsDNA) and—by assessing the extent of base-pairing with the filamentous ssDNA—identify regions of DNA that share substantial sequence similarity to the DSB site (Prentiss et al, 2015). Ejection of this extended strand allows it to anneal with the complementary DNA on the other

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Conclusion