Cooperative RecA clustering: the key to efficient homology searching

Andrew J Lee,Christoph Wälti,Jamie K Hobbs,Rajan Sharma

doi:10.1093/nar/gkx769

Andrew J Lee, Christoph Wälti + Show 2 more

Open Access

https://doi.org/10.1093/nar/gkx769

Copy DOI

Abstract

The mechanism by which pre-synaptic RecA nucleoprotein filaments efficiently locate sequence homology across genomic DNA remains unclear. Here, using atomic force microscopy, we directly investigate the intermediates of the RecA-mediated homologous recombination process and find it to be highly cooperative, involving multiple phases. Initially, the process is dominated by a rapid ‘association’ phase, where multiple filaments interact on the same dsDNA simultaneously. This cooperative nature is reconciled by the observation of localized dense clusters of pre-synaptic filaments interacting with the observed dsDNA molecules. This confinement of reactive species within the vicinity of the dsDNA, is likely to play an important role in ensuring that a high interaction rate between the nucleoprotein filaments and the dsDNA can be achieved. This is followed by a slower ‘resolution’ phase, where the synaptic joints either locate sequence homology and progress to a post-synaptic joint, or dissociate from the dsDNA. Surprisingly, the number of simultaneous synaptic joints decreases rapidly after saturation of the dsDNA population, suggesting a reduction in interaction activity of the RecA filaments. We find that the time-scale of this decay is in line with the time-scale of the dispersion of the RecA filament clusters, further emphasising the important role this cooperative phenomena may play in the RecA-facilitated homology search.

Highlights

Homologous recombination is critical in maintaining genomic integrity, repairing detrimental DNA damage that would otherwise elicit deleterious cell activity, and effecting genetic diversity during meiosis [1,2]
In order to study the intermediates formed between presynaptic filaments and double-stranded DNA (dsDNA) during the RecA-facilitated homology search, atomic force microscopy (AFM) images of the reaction were acquired periodically over the time-course of an hour
The region of homology was located 360 bp away from the 5 terminal, i.e. 85 bp away from the centre, to enable clear distinction between synaptic and the postsynaptic joints. 60-nt-long nucleoprotein filaments have been shown to be highly efficient at forming synaptic joints[30,35] and are of comparable length to those used in previous Forster resonance energy transfer (FRET)-based studies [24]

Summary

Introduction

Homologous recombination is critical in maintaining genomic integrity, repairing detrimental DNA damage that would otherwise elicit deleterious cell activity, and effecting genetic diversity during meiosis [1,2]. The binding of the incoming dsDNA into site II induces a kink in the dsDNA, disrupting the base-stacking and considerably weakening the basepairing which leads to a local opening of the dsDNA duplex in the vicinity of the encapsulated ssDNA [8,9,10,11] This process has been demonstrated to be enhanced where the stability of the duplex is already compromised [12], and is likely critically dependent upon spontaneous fluctuations in the DNA conformation, commonly referred to as DNA breathing, in line with many sequence specific DNA binding proteins that require transient access to a single strand of the duplex [13,14]

Methods

Results

Conclusion