Human BRCA2 protein promotes RAD51 filament formation on RPA-covered single-stranded DNA

Liu, Jie; Doty, Tammy; Gibson, Bryan A.; Heyer, Wolf Dietrich

doi:10.1038/nsmb.1904

Cited by 339 publications

(310 citation statements)

References 19 publications

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“…However, Srs2 contains a BRC repeat variant (BRCv, a.a.836–860). Srs2‐BRCv structurally resembles the BRC repeat of the human tumour suppressor BRCA2 (Islam et al , 2012) which mediates BRCA2–RAD51 interaction, thereby promoting recruitment of RAD51 to DSBs and regulation of RAD51 recombinase activity (Jensen et al , 2010; Liu et al , 2010; Thorslund et al , 2010). Srs2‐BRCv also promotes Srs2–Rad51 interaction in vitro (Islam et al , 2012).…”

Section: Discussionmentioning

confidence: 99%

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

et al. 2017

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Cells use homology‐dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology‐based mechanisms involves nuclease‐dependent DNA end resection, which generates long tracts of single‐stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re‐synthesis of resected DNA. We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA. As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re‐synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single‐stranded gap and terminate further resection.

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Section: Discussionmentioning

confidence: 99%

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

et al. 2017

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“…However, only filaments formed on the ssDNA substrates are productive in homologous pairing and DNA strand exchange (41). The recombination mediator BRCA2 is known to promote a bias in binding affinity favoring the ssDNA (36,37,42). We investigated the effect of RAD51 phosphorylation on the RAD51-dsDNA and RAD51-ssDNA interactions by incubating the RAD51 phosphomimetic mutants with linearized ϕX174 dsDNA or ϕX174 ssDNA and analyzing the electrophoretic mobility-shift assay (EMSA).…”

Section: Rad51 Phosphomimetic Mutants Efficiently Overcome the Kineticmentioning

confidence: 99%

“…While processing DSBs, the ssDNA produced by the endresection machinery is immediately bound by RPA, preventing secondary structure formation (35). The bound RPA can be replaced by RAD51 nucleoprotein filament in a reaction facilitated by the recombination mediator BRCA2 (10,36,37). The in vitro reconstituted DNA strand exchange reaction requires RPA (30,38), which removes secondary structures in the ssDNA (31,39) and, as the reaction progresses, sequesters the displaced ssDNA, thereby preventing nonproductive RAD51 nucleoprotein complexes.…”

Section: Rad51 Phosphomimetic Mutants Efficiently Overcome the Kineticmentioning

confidence: 99%

Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics

Subramanyam

Ismail

Bhattacharya

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The DNA strand exchange protein RAD51 facilitates the central step in homologous recombination, a process fundamentally important for accurate repair of damaged chromosomes, restart of collapsed replication forks, and telomere maintenance. The active form of RAD51 is a nucleoprotein filament that assembles on single-stranded DNA (ssDNA) at the sites of DNA damage. The c-Abl tyrosine kinase and its oncogenic counterpart BCR-ABL fusion kinase phosphorylate human RAD51 on tyrosine residues 54 and 315. We combined biochemical reconstitutions of the DNA strand exchange reactions with total internal reflection fluorescence microscopy to determine how the two phosphorylation events affect the biochemical activities of human RAD51 and properties of the RAD51 nucleoprotein filament. By mimicking RAD51 tyrosine phosphorylation with a nonnatural amino acid, p-carboxymethyl-L-phenylalanine (pCMF), we demonstrated that Y54 phosphorylation enhances the RAD51 recombinase activity by at least two different mechanisms, modifies the RAD51 nucleoprotein filament formation, and allows RAD51 to compete efficiently with ssDNA binding protein RPA. In contrast, Y315 phosphorylation has little effect on the RAD51 activities. Based on our work and previous cellular studies, we propose a mechanism underlying RAD51 activation by c-Abl/BCR-ABL kinases.RAD51 recombinase | c-Abl tyrosine kinase | homologous recombination | single-molecule total internal reflection fluorescence microscopy | phosphorylation T he DNA in the human genome is constantly subjected to damage. This damage is a byproduct of normal cellular metabolism, exposure to radiation, and environmental mutagens (1). Homologous recombination (HR) and the pathways that use the machinery of HR are responsible for the accurate repair of the most deleterious DNA lesions, including double-stranded DNA breaks (DSBs), interstrand DNA cross-links, and damaged replication forks, and thereby contribute to maintenance of the stable genome (2-5). HR also plays an important role in telomere maintenance (6). HR, a process highly conserved throughout evolution, is carried out through a precisely coordinated and tightly regulated series of events. The key step in HR is the assembly of a RecA-family recombinase (phage UvsX, bacterial RecA, archaeal RadA, or eukaryotic RAD51) onto resected single-stranded DNA (ssDNA). The recombinase forms a nucleoprotein filament that then invades homologous duplex DNA, resulting in a displacement loop structure that can be used as a primer for synthesis using the intact duplex as a template. Similar to its bacterial and yeast homologs, the human RAD51 binds ssDNA in an ATP-dependent manner (7). The nucleoprotein filament formed by the ATP-bound RAD51 is arranged such that each RAD51 monomer binds three nucleotides, forming the pairing unit in these reactions (8). Beyond these basic attributes, the characteristics of human RAD51 protein differ significantly from its archaeal, bacterial, and yeast homologs.The critical role of HR requires all steps of this process ...

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“…BRCA2 | DMC1 | RAD51 | mediator | meiosis T he breast cancer susceptibility protein 2, BRCA2, regulates RAD51-mediated homologous recombination (HR) (1)(2)(3). Both RAD51, a DNA strand exchange protein, and its meiotic counterpart, DMC1 (disrupted meiotic cDNA 1 or DNA meiotic recombinase 1), promote HR through the formation of a nucleoprotein filament on ssDNA (4).…”

mentioning

confidence: 99%

BRCA2 regulates DMC1-mediated recombination through the BRC repeats

Martinez

Nicolai

Kim

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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In somatic cells, BRCA2 is needed for RAD51-mediated homologous recombination. The meiosis-specific DNA strand exchange protein, DMC1, promotes the formation of DNA strand invasion products (joint molecules) between homologous molecules in a fashion similar to RAD51. BRCA2 interacts directly with both human RAD51 and DMC1; in the case of RAD51, this interaction results in stimulation of RAD51-promoted DNA strand exchange. However, for DMC1, little is known regarding the basis and functional consequences of its interaction with BRCA2. Here we report that human DMC1 interacts directly with each of the BRC repeats of BRCA2, albeit most tightly with repeats 1-3 and 6-8. However, BRC1-3 bind with higher affinity to RAD51 than to DMC1, whereas BRC6-8 bind with higher affinity to DMC1, providing potential spatial organization to nascent filament formation. With the exception of BRC4, each BRC repeat stimulates joint molecule formation by DMC1. The basis for this stimulation is an enhancement of DMC1-ssDNA complex formation by the stimulatory BRC repeats. Lastly, we demonstrate that full-length BRCA2 protein stimulates DMC1-mediated DNA strand exchange between RPAssDNA complexes and duplex DNA, thus identifying BRCA2 as a mediator of DMC1 recombination function. Collectively, our results suggest unique and specialized functions for the BRC motifs of BRCA2 in promoting homologous recombination in meiotic and mitotic cells.T he breast cancer susceptibility protein 2, BRCA2, regulates RAD51-mediated homologous recombination (HR) (1-3). Both RAD51, a DNA strand exchange protein, and its meiotic counterpart, DMC1 (disrupted meiotic cDNA 1 or DNA meiotic recombinase 1), promote HR through the formation of a nucleoprotein filament on ssDNA (4). This filament finds and invades a homologous template, resulting in a DNA strand invasion product called a joint molecule or a displacement-loop (D-loop). The joint molecule provides a primer template for the new DNA synthesis required to repair the DNA double strand break (DSB).The first evidence implicating BRCA2 in meiosis came from studies in Ustilago maydis, where strains lacking the BRCA2 ortholog, Brh2, resulted in absence of meiotic products (5). Shortly thereafter, mouse BRCA2 was inferred to coordinate the activities of RAD51 and DMC1 (6). The first direct interaction between BRCA2 and DMC1 was observed in plants (7) and later in humans (8). In the plant, Arabidopsis thaliana, the interaction between Brca2 and Dmc1 was mapped to the BRC repeats (9), a highly conserved motif comprising a sequence of ∼35 amino acids that is present at least once in all BRCA2-like proteins (10). In humans, BRCA2 contains eight BRC repeats that bind with different affinities to RAD51, and they segregate into two functional classes (11). Within a BRC repeat, two motifs that bind RAD51 have been identified: one comprising the consensus sequence FxxA that mimics the oligomerization interface (12) and contacts the catalytic domain of RAD51; the other binding module comprises the alpha-helical region o...

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Human BRCA2 protein promotes RAD51 filament formation on RPA-covered single-stranded DNA

Cited by 339 publications

References 19 publications

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics

BRCA2 regulates DMC1-mediated recombination through the BRC repeats

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