Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2

Esashi, Fumiko; Galkin, Vitold E.; Xiong, Yong; Egelman, Edward H.; West, Stephen C.

doi:10.1038/nsmb1245

Cited by 239 publications

(242 citation statements)

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“…Recent studies identified two peptide fragments of BRCA2 that can act to promote RAD51 filaments (28,29). These peptides seem to bind an interface created by two adjacent RAD51 monomers.…”

Section: Discussionmentioning

confidence: 99%

A chemical compound that stimulates the human homologous recombination protein RAD51

Jayathilaka

Sheridan

Bold

et al. 2008

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

127

144

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RAD51 and other members of the RecA family of strand exchange proteins assemble on ssDNA to form presynaptic filaments, which carry out the central steps of homologous recombination. A microplate-based assay was developed for high-throughput measurement of hRAD51 filament formation on ssDNA. With this method, a 10,000 compound library was screened, leading to the identification of a small molecule (RS-1) that enhances hRAD51 binding in a wide range of biochemical conditions. Salt titration experiments showed that RS-1 can enhance filament stability. Ultrastructural analysis of filaments formed on ssDNA showed that RS-1 can increase both protein-DNA complex lengths and the pitch of helical filament turns. RS-1 stimulated hRAD51-mediated homologous strand assimilation (D-loop) activity by at least 5-to 11-fold, depending on the condition. This D-loop stimulation occurred even in the presence of Ca 2؉ or adenylyl-imidodiphosphate, indicating that the mechanism of stimulation was distinct from that conferred by Ca 2؉ and/or inhibition of ATPase. No D-loop activity was observed in the absence of a nucleotide triphosphate cofactor, indicating that the compound does not substitute for this requirement. These results indicate that RS-1 enhances the homologous recombination activity of hRAD51 by promoting the formation of active presynaptic filaments. Cell survival assays in normal neonatal human dermal fibroblasts demonstrated that RS-1 promotes a dose-dependent resistance to the cross-linking chemotherapeutic drug cisplatin. Given that RAD51-dependent recombination is a major determinant of cisplatin resistance, RS-1 seems to function in vivo to stimulate homologous recombination repair proficiency. RS-1 has many potential applications in both research and medical settings.cross-linking chemotherapy ͉ DNA repair ͉ high-throughput screen ͉ recombinase ͉ strand exchange H omologous recombination (HR) has multiple roles in DNA repair, including the repair of double strand breaks (DSBs) and recovery from the replication-blocking lesions formed by DNA cross-linking agents. HR repairs DSBs by locating a homologous stretch of DNA and replicating the missing genetic information from this homologous template. In contrast to DSB repair by nonhomologous end joining, HR repair generally occurs without mutations. Because of this, HR repair is critically important in the maintenance of genomic stability (reviewed in ref. 1). The proposed mechanism for this pathway begins with 5Ј to 3Ј nuclease activity at the DSB, resulting in a 3Ј singlestranded tail. The tail is coated by replication protein A, which is subsequently replaced by a helical filament of RAD51 protein. This displacement of replication protein A by RAD51 seems to be controlled by a number of mediator proteins, which include BRCA2, RAD52, and RAD51 paralogue complexes (2-5). The RAD51-coated 3Ј tail then locates and invades a homologous template of dsDNA. After invasion, templated DNA synthesis initiated at 3Ј ends leads to formation of branched DNA intermediates, which ...

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“…Recent studies identified two peptide fragments of BRCA2 that can act to promote RAD51 filaments (28,29). These peptides seem to bind an interface created by two adjacent RAD51 monomers.…”

Section: Discussionmentioning

confidence: 99%

A chemical compound that stimulates the human homologous recombination protein RAD51

Jayathilaka

Sheridan

Bold

et al. 2008

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

127

144

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“…It has been demonstrated that both BRC3 and BRC4 interact with the RAD51 core domain (a region of RAD51 lacking the N-terminal domain), suggesting that some aspect of the core represents the site of interaction (Wong et al, 1997;Esashi et al, 2007). Further insight into this interaction was provided when the structure of a protein comprising BRC4 fused to the RAD51 core was solved by X-ray crystallography.…”

Section: Binding and Regulation Of Rad51 By The Brc Motifs Of Brca2mentioning

confidence: 99%

“…Due to the availability of the BRC4-RAD51 core structure, many studies have focused on a peptide corresponding to BRC4 itself. The BRC4 peptide was found to disrupt RAD51 filaments, presumably by binding RAD51 monomers in solution, thereby shifting the equilibrium towards BRC4-RAD51 heterodimer formation (Davies and Pellegrini, 2007;Esashi et al, 2007). However, this disruptive effect was only observed when BRC4 was present in molar excess compared to RAD51, and at lower concentrations of BRC4 it was found to associate with RAD51 filaments formed on dsDNA in the presence of the non-hydrolysable ATP analogue 5 0 -adenylyl-b, g-imidodiphosphate (AMP-PNP) (Galkin et al, 2005).…”

Section: Regulation Of Recombination By Brca2 T Thorslund and Sc Westmentioning

confidence: 99%

“…Using synthetic peptides corresponding to the TR2 region, and mutant derivatives of RAD51 protein, it was shown that TR2 interacts specifically with multimeric RAD51 (filaments or rings) but cannot bind RAD51 monomers (Davies and Pellegrini, 2007;Esashi et al, 2007). Furthermore, TR2 was found to stabilize RAD51 filaments from the disruptive actions of BRC4, leading to the suggestion that the C-terminal domain of BRCA2 plays a positive role in the establishment of functional RAD51 nucleoprotein filaments.…”

Section: Stabilization Of Rad51 Nucleoprotein Filaments By Brca2 C-tementioning

confidence: 99%

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BRCA2: a universal recombinase regulator

2007

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Homologous recombination has a dual role in eukaryotic organisms. Firstly, it is responsible for the creation of genetic variability during meiosis by directing the formation of reciprocal crossovers that result in random combinations of alleles and traits. Secondly, in mitotic cells, it maintains the integrity of the genome by promoting the faithful repair of DNA double-strand breaks (DSBs). In vertebrates, it therefore plays a key role in tumour avoidance. Mutations in the tumour suppressor protein BRCA2 are associated with predisposition to breast and ovarian cancers, and loss of BRCA2 function leads to genetic instability. BRCA2 protein interacts directly with the RAD51 recombinase and regulates recombinationmediated DSB repair, accounting for the high levels of spontaneous chromosomal aberrations seen in BRCA2-defective cells. Recent observations indicate that BRCA2 also plays a critical role in meiotic recombination, this time through direct interactions with the meiosis-specific recombinase DMC1. The interactions of BRCA2 with RAD51 and DMC1 lead us to suggest that the BRCA2 tumour suppressor is a universal regulator of recombinase actions.

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“…Group III consists of FANCD1 (BRCA2), FANCN (PALB2), and FANCJ (BRIP1), which do not play a role in FANCD2 monoubiquitination. BRCA2 regulates formation of RAD51 nucleoprotein filaments during homologous recombination (14,15), PALB2 is necessary for the correct association of BRCA2 with chromatin (16), and BRIP1 is a BRCA1-associated DNA helicase that contributes to homologous recombination and cross-link repair (17,18).…”

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confidence: 99%

Remodeling of DNA replication structures by the branch point translocase FANCM

Gari

Décaillet²,

Delannoy³

et al. 2008

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

209

178

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Fanconi anemia (FA) is a genetically heterogeneous chromosome instability syndrome associated with congenital abnormalities, bone marrow failure, and cancer predisposition. Eight FA proteins form a nuclear core complex, which promotes tolerance of DNA lesions in S phase, but the underlying mechanisms are still elusive. We reported recently that the FA core complex protein FANCM can translocate Holliday junctions. Here we show that FANCM promotes reversal of model replication forks via concerted displacement and annealing of the nascent and parental DNA strands. Fork reversal by FANCM also occurs when the lagging strand template is partially single-stranded and bound by RPA. The combined fork reversal and branch migration activities of FANCM lead to extensive regression of model replication forks. These observations provide evidence that FANCM can remodel replication fork structures and suggest a mechanism by which FANCM could promote DNA damage tolerance in S phase.fanconi anemia ͉ replication fork A variety of structural and chemical alterations in DNA can hinder the progression of replication forks and precipitate the formation of gross chromosomal rearrangements. These hurdles impose distinct structural constraints in the template DNA, which elicit the action of diverse lesion bypass or lesion tolerance pathways (1, 2). Covalent links between complementary DNA strands constitute a unique challenge to replicating cells, because they preclude strand separation and, hence, completely block fork progression. In mammalian cells, the repair of DNA interstrand cross-links (ICLs) is thought to take place during S phase (3). The exact mechanism of repair is unknown, but it seems to involve the interplay of different pathways, with the homologous recombination machinery, translesion DNA polymerases, and the Fanconi anemia (FA) pathway all being required for ICL tolerance (4).FA is a genetically heterogeneous inherited disorder, which combines congenital abnormalities, bone marrow failure, and a marked cancer predisposition (5-8). FA cells are prone to spontaneous and damage-induced chromosomal aberrations and are notoriously hypersensitive to DNA interstrand cross-linking agents. FA proteins can be classified into three groups (8). Group I includes FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM. These eight FA proteins form a nuclear core complex (9-11) whose integrity is required for the conjugation of a ubiquitin moiety to the group II proteins, FANCI and FANCD2 (12,13). Group III consists of FANCD1 (BRCA2), FANCN (PALB2), and FANCJ (BRIP1), which do not play a role in FANCD2 monoubiquitination. BRCA2 regulates formation of RAD51 nucleoprotein filaments during homologous recombination (14, 15), PALB2 is necessary for the correct association of BRCA2 with chromatin (16), and BRIP1 is a BRCA1-associated DNA helicase that contributes to homologous recombination and cross-link repair (17, 18).The FA core complex protein FANCM can specifically bind to model replication forks and Holliday junctions and move the...

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Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2

Cited by 239 publications

References 28 publications

A chemical compound that stimulates the human homologous recombination protein RAD51

A chemical compound that stimulates the human homologous recombination protein RAD51

BRCA2: a universal recombinase regulator

Remodeling of DNA replication structures by the branch point translocase FANCM

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