The Saccharomyces cerevisiae Shu complex, consisting of Shu1, Shu2, Csm2 and Psy3, promotes error-free homologous recombination (HR) by an unknown mechanism. Recent structural analysis of two Shu proteins, Csm2 and Psy3, has revealed that these proteins are Rad51 paralogues and mediate DNA binding of this complex. We show in vitro that the Csm2–Psy3 heterodimer preferentially binds synthetic forked DNA or 3′-DNA overhang substrates resembling structures used during HR in vivo. We find that Csm2 interacts with Rad51 and the Rad51 paralogues, the Rad55–Rad57 heterodimer and that the Shu complex functions in the same epistasis group as Rad55–Rad57. Importantly, Csm2’s interaction with Rad51 is dependent on Rad55, whereas Csm2’s interaction with Rad55 occurs independently of Rad51. Consistent with the Shu complex containing Rad51 paralogues, the methyl methanesulphonate sensitivity of Csm2 is exacerbated at colder temperatures. Furthermore, Csm2 and Psy3 are needed for efficient recruitment of Rad55 to DNA repair foci after DNA damage. Finally, we observe that the Shu complex preferentially promotes Rad51-dependent homologous recombination over Rad51-independent repair. Our data suggest a model in which Csm2–Psy3 recruit the Shu complex to HR substrates, where it interacts with Rad51 through Rad55–Rad57 to stimulate Rad51 filament assembly and stability, promoting error-free repair.
The conserved budding yeast Rad51 paralogues, including Rad55, Rad57, Csm2 and Psy3 are indispensable for homologous recombination (HR)-mediated chromosome damage repair. Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2. Here we show that Rad55 bridges an interaction between Csm2 with Rad51 and Rad52 and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55–Rad57 and Rad52 to promote nucleation of Rad51 on single-stranded DNA pre-occupied by replication protein A (RPA). The csm2–F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo. Our results reveal that Rad55–Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.
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 RAD-51 paralogue was discovered in C. elegans and its in vitro characterization has demonstrated a new function for the worm RAD-51 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.
Here, we investigate the role of the budding yeast Shu complex in promoting homologous recombination (HR) upon replication fork damage. We recently found that the Shu complex stimulates Rad51 filament formation during HR through its physical interactions with Rad55-Rad57. Unlike other HR factors, Shu complex mutants are primarily sensitive to replicative stress caused by MMS and not to more direct DNA breaks. Here, we uncover a novel role for the Shu complex in the repair of specific MMS-induced DNA lesions and elucidate the interplay between HR and translesion DNA synthesis. We find that the Shu complex promotes high-fidelity bypass of MMS-induced alkylation damage, such as N3-methyladenine, as well as bypassing the abasic sites generated after Mag1 removes N3-methyladenine lesions. Furthermore, we find that the Shu complex responds to ssDNA breaks generated in cells lacking the abasic site endonucleases. At each lesion, the Shu complex promotes Rad51-dependent HR as the primary repair/tolerance mechanism over error-prone translesion DNA polymerases. Together, our work demonstrates that the Shu complex's promotion of Rad51 pre-synaptic filaments is critical for high-fidelity bypass of multiple replication-blocking lesion.
The Saccharomyces cerevisiae Shu2 protein is an important regulator of Rad51, which promotes homologous recombination (HR). Shu2 functions in the Shu complex with Shu1 and the Rad51 paralogs Csm2 and Psy3. Shu2 belongs to the SWS1 protein family, which is characterized by its SWIM domain (CXC...X n ...CXH), a zinc-binding motif. In humans, SWS1 interacts with the Rad51 paralog SWSAP1. Using genetic and evolutionary analyses, we examined the role of the Shu complex in mitotic and meiotic processes across eukaryotic lineages. We provide evidence that the SWS1 protein family contains orthologous genes in early-branching eukaryote lineages (e.g., Giardia lamblia), as well as in multicellular eukaryotes including Caenorhabditis elegans and Drosophila melanogaster. Using sequence analysis, we expanded the SWIM domain to include an invariant alanine three residues after the terminal CXH motif (CXC. . .X n . . .CXHXXA). We found that the SWIM domain is conserved in all eukaryotic orthologs, and accordingly, in vivo disruption of the invariant residues within the canonical SWIM domain inhibits DNA damage tolerance in yeast and protein-protein interactions in yeast and humans. Furthermore, using evolutionary analyses, we found that yeast and Drosophila Shu2 exhibit strong coevolutionary signatures with meiotic proteins, and in yeast, its disruption leads to decreased meiotic progeny. Together our data indicate that the SWS1 family is an ancient and highly conserved eukaryotic regulator of meiotic and mitotic HR.KEYWORDS DNA repair; Shu complex; budding yeast; evolutionary rate covariation; homologous recombination H OMOLOGOUS recombination (HR) is an error-free DNA repair pathway that is essential both in mitotic cells to ensure DNA stability and in meiotic cells for faithful chromosome segregation. HR begins with double-strand break (DSB) formation and DNA end processing that give rise to 39 singlestranded DNA (ssDNA) overhangs (Heyer et al. 2010). A key step in HR is the formation of RecA-like filaments on these ssDNA ends. During mitosis in the budding yeast Saccharomyces cerevisiae, the RecA-like protein Rad51 coats the ssDNA, whereas during meiosis, both Rad51 and another RecA-like protein, Dmc1, form on ssDNA ends (Lin et al. 2006;Heyer et al. 2010). Formation of RecA-like filaments on the DNA is essential for the homology search and strand-invasion steps that define HR. Therefore, regulation of RecA filament formation is critical for accurate repair of DNA damage and chromosome segregation. Given their importance, both Rad51 and Dmc1 are extremely well-conserved descendants of the archaeal protein RADA (Diruggiero et al. 1999;Komori et al. 2000;Lin et al. 2006;Chintapalli et al. 2013).There are many proteins that both promote Rad51 filament formation and disassemble inappropriate filaments. Interestingly, in many organisms, the proteins that stabilize Rad51 filaments themselves share structural homology with Rad51 and evolved from the archaeal RADB homolog (Lin et al. 2006). In humans, these Rad51 paralogs...
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