Single-molecule studies illuminate the function of RAD51 paralogs.
TL;DR: Roy et al. as mentioned in this paper demonstrated that the yeast and nematode RAD51 paralog complexes function as chaperones to promote the assembly of the RAD51 nucleoprotein filament on RPA-coated ssDNA.
About: This article is published in Molecular Cell.The article was published on 2021-03-04. It has received 2 citations till now.
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TL;DR: In this article, the authors describe recent advances in RAD51 control mechanisms obtained specifically using fluorescence-based single-molecule techniques, and describe how modulators control filament dynamics is at the heart of current research efforts.
3 citations
TL;DR: In this paper, Fang et al. showed that FANCD2 inhibits fork degradation in two ways: 1) it inhibits DNA2 nuclease activity by directly binding to DNA2 and 2) independent of dimerization with FANCI, Fang2 itself stabilizes RAD51 filaments to inhibit various nucleases, including DNA2.
Abstract: Summary FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea induced stalled forks. The mechanisms of the fork protection are not well studied. Here, we purified FANCD2 to study how FANCD2 regulates DNA resection at stalled forks. In vitro, we showed that FANCD2 inhibits fork degradation in two ways: 1) it inhibits DNA2 nuclease activity by directly binding to DNA2. 2) independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit various nucleases, including DNA2. More unexpectedly, FANCD2 acts as a RAD51 mediator to stimulate the strand exchange activity of RAD51, and does so by enhancing ssDNA binding of RAD51. Our work biochemically explains mechanisms by which FANCD2 protects stalled forks and further provides a simple molecular explanation for genetic interactions between FANCD2 and the BRCA2 mediator.
2 citations
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TL;DR: The purification of full-length BRCA2 is reported and it is shown that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA).
Abstract: Mutation of the breast cancer susceptibility gene, BRCA2, leads to breast and ovarian cancers. Mechanistic insight into the functions of human BRCA2 has been limited by the difficulty of isolating this large protein (3,418 amino acids). Here we report the purification of full-length BRCA2 and show that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). BRCA2 acts by targeting RAD51 to ssDNA over double-stranded DNA, enabling RAD51 to displace replication protein-A (RPA) from ssDNA and stabilizing RAD51-ssDNA filaments by blocking ATP hydrolysis. BRCA2 does not anneal ssDNA complexed with RPA, implying it does not directly function in repair processes that involve ssDNA annealing. Our findings show that BRCA2 is a key mediator of homologous recombination, and they provide a molecular basis for understanding how this DNA repair process is disrupted by BRCA2 mutations, which lead to chromosomal instability and cancer.
593 citations
TL;DR: In this article, a stable heterodimer of Rad51, Rad55, and Rad57 was found to exist as a stable product with a dissociation constant of <2 x 10−10 M.
Abstract: Saccharomyces cerevisiae RAD51, RAD55, and RAD57 genes, required for genetic recombination and DNA double-strand-break repair, encode proteins homologous to one another and to the Escherichia coli RecA protein. Rad51 protein catalyzes the DNA strand-exchange reaction with a dependence on ATP and on the heterotrimeric single-strand DNA (ssDNA) binding factor replication protein A (RPA). By several independent criteria, RAD55- and RAD57-encoded products are shown here to exist as a stable heterodimer, with a dissociation constant of <2 x 10(-10) M. In strand exchange, the reaction proceeds efficiently if RPA is incorporated after nucleation of Rad51 onto ssDNA, but if RPA is present during the nucleation phase, as is likely the case in vivo, the amount of strand-exchange products becomes relatively insignificant. Inclusion of the Rad55-Rad57 heterodimer with Rad51 and RPA results in a marked stimulation of strand exchange, providing evidence for a role of the Rad55-Rad57 heterodimer in overcoming the inhibitory effect of RPA.
497 citations
TL;DR: The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review.
Abstract: Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of the RecA/RAD51 filament, which promotes the homology search. On finding homology, the nucleoprotein filament catalyzes exchange of DNA strands to form a joint molecule. Recombination is controlled by regulating the fate of both RecA/RAD51 filaments and DNA pairing intermediates. Finally, intermediates that mature into Holliday structures are disjoined by either nucleolytic resolution or topological dissolution.
365 citations
TL;DR: Five Rad51-like proteins, referred to as Rad51 paralogs, have been described in vertebrates and it is shown that two of them, Rad51B and Rad51C, are associated in a stable complex that has ssDNA binding and ssDNA-stimulated ATPase activities.
Abstract: Five Rad51-like proteins, referred to as Rad51 paralogs, have been described in vertebrates. We show that two of them, Rad51B and Rad51C, are associated in a stable complex. Rad51B–Rad51C complex has ssDNA binding and ssDNA-stimulated ATPase activities. We also examined the functional interaction of Rad51B–Rad51C with Rad51 and RPA. Even though RPA enhances Rad51-catalyzed DNA joint formation via removal of secondary structure in the ssDNA substrate, it can also compete with Rad51 for binding to the substrate, leading to suppressed reaction efficiency. The competition by RPA for substrate binding can be partially alleviated by Rad51B–Rad51C. This recombination mediator function of Rad51B–Rad51C is likely required for the assembly of the Rad51-ssDNA nucleoprotein filament in vivo.
242 citations
TL;DR: It is proposed that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55–Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination.
Abstract: Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyses the signature reactions of recombination, homology search and DNA strand invasion. Eukaryotes also possess Rad51 paralogues, whose exact role in recombination remains to be defined. Here we show that the Saccharomyces cerevisiae Rad51 paralogues, the Rad55-Rad57 heterodimer, counteract the antirecombination activity of the Srs2 helicase. The Rad55-Rad57 heterodimer associates with the Rad51-single-stranded DNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51-Rad55-Rad57 co-filament resists disruption by the Srs2 antirecombinase by blocking Srs2 translocation, involving a direct protein interaction between Rad55-Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogues in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 antirecombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55-Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogues, which are known to be involved in cancer predisposition and human disease.
181 citations