Non-enzymatic roles of human RAD51 at stalled replication forks

Ralph W Weichselbaum,Douglas K Bishop,Yuen-Ling Chan,Jennifer M Mason

doi:10.1038/s41467-019-12297-0

Ralph W Weichselbaum, Douglas K Bishop + Show 2 more

Open Access

https://doi.org/10.1038/s41467-019-12297-0

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Abstract

The central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Here, we use a separation-of-function allele of RAD51 that retains DNA binding, but not D-loop activity, to reveal mechanistic aspects of RAD51’s roles in the response to replication stress. Here, we find that cells lacking RAD51’s enzymatic activity protect replication forks from MRE11-dependent degradation, as expected from previous studies. Unexpectedly, we find that RAD51’s strand exchange activity is not required to convert stalled forks to a form that can be degraded by DNA2. Such conversion was shown previously to require replication fork regression, supporting a model in which fork regression depends on a non-enzymatic function of RAD51. We also show RAD51 promotes replication restart by both strand exchange-dependent and strand exchange-independent mechanisms.

Highlights

The central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress
Previous studies have suggested that stabilization of RAD51 filaments is sufficient to protect from MRE11-dependent degradation[7,13]
This study utilized the RAD51-K133R mutant that is defective for homologous recombination (HR)-mediated repair, but has robust strand exchange activity precluding the use of this mutant to definitively determine if RAD51 DNA binding activity alone is sufficient to protect from MRE11-dependent degradation[26,27]

Summary

Introduction

The central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Once RAD51 filaments form on tracts of ssDNA, the protein alters the structure of the ssDNA, allowing the nucleoprotein filament to catalyze a homology search to identify an identical or nearly identical sequence in duplex DNA, and carry out exchange of the bound ssDNA strand with the like strand of the homologous duplex[3]. In this way, the homology search and strand exchange activity of RAD51 acts to form a homologous joint between a broken chromatid and its intact sister chromatid, leading to accurate repair of the DSB. Nascent DNA degradation occurs in cells lacking proteins required to load

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