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Review
. 2016 Oct;94(5):407-418.
doi: 10.1139/bcb-2016-0012. Epub 2016 Mar 31.

Novel Insights Into RAD51 Activity and Regulation During Homologous Recombination and DNA Replication

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Free PMC article
Review

Novel Insights Into RAD51 Activity and Regulation During Homologous Recombination and DNA Replication

Stephen K Godin et al. Biochem Cell Biol. .
Free PMC article

Abstract

In this review we focus on new insights that challenge our understanding of homologous recombination (HR) and Rad51 regulation. Recent advances using high-resolution microscopy and single molecule techniques have broadened our knowledge of Rad51 filament formation and strand invasion at double-strand break (DSB) sites and at replication forks, which are one of most physiologically relevant forms of HR from yeast to humans. Rad51 filament formation and strand invasion is regulated by many mediator proteins such as the Rad51 paralogues and the Shu complex, consisting of a Shu2/SWS1 family member and additional Rad51 paralogues. Importantly, a novel RAD51 paralogue was discovered in Caenorhabditis elegans, and its in vitro characterization has demonstrated a new function for the worm RAD51 paralogues during HR. Conservation of the human RAD51 paralogues function during HR and repair of replicative damage demonstrate how the RAD51 mediators play a critical role in human health and genomic integrity. Together, these new findings provide a framework for understanding RAD51 and its mediators in DNA repair during multiple cellular contexts.

Keywords: DNA replication; RAD51; Rad51 paralogues; Shu complex; complexe Shu; homologous recombination; paralogues de Rad51; recombinaison homologue; réplication d’ADN.

Figures

Figure 1
Figure 1
The different pathways utilized during homologous recombination. Protein names in black refer to budding yeast and are used in the legend, while red names refer to human proteins. All pathways begin with recognition of the DSB, followed by resection mediated by the MRX complex in conjunction with Sae2, Exo1, and Sgs1-Dna2 to generated extended 3′ ssDNA overhangs. These overhangs are immediately coated by the ssDNA binding protein complex RPA. In order for HR to proceed, Rad51 filaments are induced to form on the ssDNA overhangs by the activity of Rad52, the Rad51 paralogues, and the Shu complex. Rad51 filaments, in conjunction with Rad54 and Rdh54, carry out the critical homology search and strand invasion steps of HR. The strand invasion step is the last common step for all the HR pathways modeled here. During SDSA, the strand invasion product is extended past the break-site before being disassembled and reannealed to the other side of the break, generating a non-crossover repair product. In BIR, the strand invasion product becomes a full replication fork and can progress to the end of the chromosome, creating widespread loss-of-heterozygosity. Alternatively, the second end of the DSB can be captured to generate a double Holliday junction (Center). This double Holliday junction can be cleaved by various nucleases to generate both non-crossover and crossover events as depicted on the left and right respectively. The double Holliday junction can also be dissolved by Sgs1-Top3-Rmi1 to yield non-crossover events. Dashed lines refer to DNA synthesized during HR.
Figure 2
Figure 2
A schematic of the major HR proteins discussed. Homologues in five eukaryotic lineages are shown, and the Rad51 paralogues and the Shu complex, which are discussed in detail in this review, are drawn to show the known complex members in each species.
Figure 3
Figure 3
A schematic highlighting three potential methods to restart a stalled replication fork in budding yeast. In this model, a blocking lesion, indicated by a red star, stalls replication behind the replication fork. This lesion can be bypassed by error-prone translesion polymerases (Top right; TLS), or it can be bypassed using high-fidelity HR (Bottom). Two potential models of how HR can progress at a damaged replication fork are shown. In the model on the left, the ssDNA gap at the replication fork serves as the template for HR. In the right-hand model, the newly synthesized, blocked DNA strand serves as the template. In both pathways, ssDNA is RPA-coated before Rad51 filaments are formed by the activities of Rad52, Rad55-Rad57, and the Shu complex. At the same time, illegitimate HR is inhibited by the anti-recombinase Srs2. Rad51-coated DNA is able to invade the undamaged, newly-synthesized sister chromatid by the activity of Rad54 and Rdh54. This allows extension by different DNA polymerases past the blocking lesion. Finally, both the gap invasion and end invasion pathways can either be dissolved into a non-crossover product (Bottom left) or result in the formation of a single or double Holliday junction (Bottom right), which is then dissolved by Sgs1-Top3-Rmi1.
Figure 4
Figure 4
Hypothetical models for how the yeast Rad51 paralogues could associate with the Rad51-Rad52-RPA filament during HR [adapted from (Gibb et al. 2014)]. A DSB is recognized and resected to generate 3′ ssDNA overhangs, which are immediately coated by the ssDNA binding complex RPA. Rad52 then binds the RPA-coated ssDNA and stabilizes a patch of RPA on the ssDNA. Next, the Rad51 paralogues, Rad55-Rad57, form a higher order ensemble with Rad52 and the Shu complex to stimulate Rad51 filament formation. Although filaments of Rad52-RPA coat the Rad51 nucleoprotein filament, whether Rad55-Rad57 and the Shu complex exhibit similar interactions with the Rad51 nucleoprotein filament remains unknown. Three hypothetical models for how the Rad51 paralogues and the Shu complex may interact with the Rad52-RPA-Rad51 nucleoprotein filament are shown. On the left, Rad55-Rad57 and the Shu complex are released after Rad51 filaments form and are not associated with the Rad52-RPA-Rad51 filament. In the middle model, Rad55-Rad57 and the Shu complex are integrated into the Rad51 nucleoprotein filament but have no additional interactions with Rad52-RPA. On the right, Rad55-Rad57 and the Shu complex interact with Rad52-RPA and together coat the Rad51 filament as a co-filament. Importantly, the middle and right-hand models are not mutually exclusive, and Rad55-Rad57 and the Shu complex could both integrate into the Rad51 filament as well as form a co-filament with Rad52-RPA.

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