High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy.

Judith M Short,Ashok R Venkitaraman,Yang Liu,Neelesh Soni,Mallur S Madhusudhan,Shaoxia Chen,Mahmud K.K Shivji

doi:10.1093/nar/gkw783

Abstract

Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses pairing with homologous double-stranded (ds) DNA. Here, we report a structure for the presynaptic human RAD51 filament at 3.5–5.0Å resolution using electron cryo-microscopy. RAD51 encases ssDNA in a helical filament of 103Å pitch, comprising 6.4 protomers per turn, with a rise of 16.1Å and a twist of 56.2°. Inter-protomer distance correlates with rotation of an α-helical region in the core catalytic domain that is juxtaposed to ssDNA, suggesting how the RAD51–DNA interaction modulates protomer spacing and filament pitch. We map Fanconi anaemia-like disease-associated RAD51 mutations, clarifying potential phenotypes. We predict binding sites on the presynaptic filament for two modules present in each BRC repeat of the BRCA2 tumour suppressor, a critical HR mediator. Structural modelling suggests that changes in filament pitch mask or expose one binding site with filament-inhibitory potential, rationalizing the paradoxical ability of the BRC repeats to either stabilize or inhibit filament formation at different steps during HR. Collectively, our findings provide fresh insight into the structural mechanism of HR and its dysregulation in human disease.

Highlights

Homologous DNA recombination (HR) to repair dsDNA breaks begins with the 5 to 3 endonucleolytic resection of DNA ends, generating a long, overhanging 3 tail that may extend for hundreds of nucleotides
Sample preparation for full-length human RAD51 assembled on ssDNA in the presence of the adenosine triphosphate (ATP) analogue, AMPPNP, with methods for electron cryo-microscopy, are described elsewhere (‘Materials and Methods’ section, Supplementary Figure S1)
The average pitch of 103Ais closer to the 94Apitch reported for the active RecA filament on ssDNA [12], than the 130Apitch reported for a mutant form of yeast Rad51 without DNA [16], and concordant with previous measurements of active filaments (Table 1)

Summary

Introduction

Homologous DNA recombination (HR) to repair dsDNA breaks (reviewed in 1) begins with the 5 to 3 endonucleolytic resection of DNA ends, generating a long, overhanging 3 tail that may extend for hundreds of nucleotides. Crystal structures have been reported for RAD51 [11] as well as its orthologues from simpler organisms, including RecA from Escherichia coli [12,13,14], RadA from Pyrococcus furiosus [15] and Rad from Saccharomyces cerevisiae [16]. These proteins spontaneously oligomerize even in the absence of DNA to form rings with 6 or more protomers, or heli-

Methods

Results

Conclusion