Sequence fingerprints in BRCA2 and RAD51: implications for DNA repair and cancer

In humans, the interactions between the breast cancer susceptibility protein, BRCA2, and the RAD51 recombinase are essential for DNA repair by homologous recombination (HR), failure of which can predispose to cancer. The interactions occur through conserved BRC repeat motifs, encoded in BRCA2, binding directly to RAD51. Here, we describe full and partial BRCA2 homologues from a wide range of eukaryotes, including Drosophila melanogaster and two Plasmodium species. The crystal structure of the human BRC4-RAD51 complex allows identification of residues that are important for protein-protein interaction, and defines interaction sequence fingerprints for the BRC repeat and for RAD51. These allow us to predict that most eukaryotic RAD51 and BRC repeat orthologues should be capable of mutual interactions. We find no evidence for the presence of BRC repeats in yeast, Archaea and bacteria, and their RAD51 orthologues do not fulfil the criteria for binding the BRC repeat. Similarly, human RAD51 paralogues, including RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1, are not predicted to bind the BRC repeat. Conservation of the BRC repeat and RAD51 sequence fingerprints across a wide range of eukaryotic species substantiates the functional significance of the BRCA2-RAD51 interactions. The idea of multiple BRC repeats with binding specificity towards RAD51 leads us to suggest a possible model for the participation of BRCA2 in RAD51 nucleoprotein filament formation.     

Role of BRCA2 in control of the RAD51 recombination and DNA repair protein

Individuals carrying BRCA2 mutations are predisposed to breast and ovarian cancers. Here, we show that BRCA2 plays a dual role in regulating the actions of RAD51, a protein essential for homologous recombination and DNA repair. First, interactions between RAD51 and the BRC3 or BRC4 regions of BRCA2 block nucleoprotein filament formation by RAD51. Alterations to the BRC3 region that mimic cancer-associated BRCA2 mutations fail to exhibit this effect. Second, transport of RAD51 to the nucleus is defective in cells carrying a cancer-associated BRCA2 truncation. Thus, BRCA2 regulates both the intracellular localization and DNA binding ability of RAD51. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis.     

A region of human BRCA2 containing multiple BRC repeats promotes RAD51-mediated strand exchange

Human BRCA2, a breast and ovarian cancer suppressor, binds to the DNA recombinase RAD51 through eight conserved BRC repeats, motifs of approximately 30 residues, dispersed across a large region of the protein. BRCA2 is essential for homologous recombination in vivo, but isolated BRC repeat peptides can prevent the assembly of RAD51 into active nucleoprotein filaments in vitro, suggesting a model in which BRCA2 sequesters RAD51 in undamaged cells, and promotes recombinase function after DNA damage. How BRCA2 might fulfill these dual functions is unclear. We have purified a fragment of human BRCA2 (BRCA2(BRC1-8)) with 1127 residues spanning all 8 BRC repeats but excluding the C-terminal DNA-binding domain (BRCA2(CTD)). BRCA2(BRC1-8) binds RAD51 nucleoprotein filaments in a ternary complex, indicating it may organize RAD51 on DNA. Human RAD51 is relatively ineffective in vitro at strand exchange between homologous DNA molecules unless non-physiological ions like NH4+ are present. In an ionic milieu more typical of the mammalian nucleus, BRCA2(BRCI-8) stimulates RAD51-mediated strand exchange, suggesting it may be an essential co-factor in vivo. Thus, the human BRC repeats, embedded within their surronding sequences as an eight-repeat unit, mediate homologous recombination independent of the BRCA2(CTD) through a previously unrecognized role in control of RAD51 activity.     

Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombinase

The breast and ovarian cancer suppressor protein BRCA2 controls the RAD51 recombinase in reactions that lead to homologous DNA recombination (HDR). BRCA2 binds RAD51 via eight conserved BRC repeat motifs of approximately 35 amino acids, each with a varying capacity to bind RAD51. BRC repeats both promote and inhibit RAD51 assembly on different DNA substrates to regulate HDR, but the structural basis for these functions is unclear. Here, we demarcate two tetrameric clusters of hydrophobic residues in the BRC repeats, interacting with distinct pockets in RAD51, and show that the co-location of both modules within a single BRC repeat is necessary for BRC–RAD51 binding and function. The two modules comprise the sequence FxxA, known to inhibit RAD51 assembly by blocking the oligomerization interface, and a previously unrecognized tetramer with the consensus sequence LFDE, which binds to a RAD51 pocket distinct from this interface. The LFDE motif is essential in BRC repeats for modes of RAD51 binding both permissive and inhibitory to RAD51 oligomerization. Targeted insertion of point mutations in RAD51 that disrupt the LFDE-binding pocket impair its assembly at DNA damage sites in living cells. Our findings suggest a model for the modular architecture of BRC repeats that provides fresh insight into the mechanisms regulating homologous DNA recombination.     

Trypanosoma brucei BRCA2 acts in antigenic variation and has undergone a recent expansion in BRC repeat number that is important during homologous recombination

Antigenic variation in Trypanosoma brucei has selected for the evolution of a massive archive of silent Variant Surface Glycoprotein (VSG) genes, which are activated by recombination into specialized expression sites. Such VSG switching can occur at rates substantially higher than background mutation and is dependent on homologous recombination, a core DNA repair reaction. A key regulator of homologous recombination is BRCA2, a protein that binds RAD51, the enzyme responsible for DNA strand exchange. Here, we show that T. brucei BRCA2 has undergone a recent, striking expansion in the number of BRC repeats, a sequence element that mediates interaction with RAD51. T. brucei BRCA2 mutants are shown to be significantly impaired in antigenic variation and display genome instability. By generating BRCA2 variants with reduced BRC repeat numbers, we show that the BRC expansion is crucial in determining the efficiency of T. brucei homologous recombination and RAD51 localization. Remarkably, however, this appears not to be a major determinant of the activation of at least some VSG genes.     

Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2

To clarify RAD51 interactions controlling homologous recombination, we report here the crystal structure of the full-length RAD51 homolog from Pyrococcus furiosus. The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA-bound filaments matching three-dimensional electron microscopy reconstructions. A polymerization motif (RAD51-PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric 'switch' promoting ATPase activity and DNA binding roles for the N-terminal domain helix-hairpin-helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P.furiosus RAD51 mutant binds BRC repeats and forms BRCA2-dependent nuclear foci in human cells in response to gamma-irradiation-induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51-PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination.     

Effect of the BRCA2 CTRD domain on RAD51 filaments analyzed by an ensemble of single molecule techniques

Homologous recombination is essential for the preservation of genome stability, thereby preventing cancer. The recombination protein RAD51 drives DNA strand exchange, which requires the assembly, rearrangement and disassembly of a RAD51 filament on DNA, coupled to ATP binding and hydrolysis. This process is facilitated and controlled by recombination mediators and accessory factors. Here, we have employed a range of single molecule techniques to determine the influence of the C-terminal RAD51 interaction domain (CTRD) of the breast cancer tumor suppressor BRCA2 on intrinsic aspects of RAD51-DNA interactions. We show that at high concentration the CTRD entangles RAD51 filaments and reduces RAD51 filament formation in a concentration dependent manner. It does not affect the rate of filament disassembly measured as the loss of fluorescent signal due to intrinsic RAD51 protein dissociation from double-stranded DNA (dsDNA). We conclude that, outside the context of the full-length protein, the CTRD does not reduce RAD51 dissociation kinetics, but instead hinders filament formation on dsDNA. The CTRDs mode of action is most likely sequestration of multiple RAD51 molecules thereby rendering them inactive for filament formation on dsDNA.     

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

The human breast cancer susceptibility gene BRCA2 is required for the regulation of RAD51-mediated homologous recombinational repair. BRCA2 interacts with RAD51 monomers, as well as nucleoprotein filaments, primarily though the conserved BRC motifs. The unrelated C-terminal region of BRCA2 also interacts with RAD51. Here we show that the BRCA2 C terminus interacts directly with RAD51 filaments, but not monomers, by binding an interface created by two adjacent RAD51 protomers. These interactions stabilize filaments so that they cannot be dissociated by association with BRC repeats. Interaction of the BRCA2 C terminus with the RAD51 filament causes a large movement of the flexible RAD51 N-terminal domain that is important in regulating filament dynamics. We suggest that interactions of the BRCA2 C-terminal region with RAD51 may facilitate efficient nucleation of RAD51 multimers on DNA and thereby stimulate recombination-mediated repair.     

Valine 1532 of human BRC repeat 4 plays an important role in the interaction between BRCA2 and RAD51

The breast cancer susceptibility protein BRCA2 is essential for recombinational DNA repair. BRCA2 specifically binds to RAD51 via eight BRC repeat motifs and delivers RAD51 to double-stranded DNA breaks. In this study, a mammalian two-hybrid assay and competitive ELISA showed that the interaction between BRC repeat 4 (BRC4) and RAD51 was strengthened by the substitution of a single BRC4 amino acid from valine to isoleucine (V1532I). However, the cancer-associated V1532F mutant exhibited very weak interaction with RAD51. This study used a comparative analysis of BRC4 between animal species to identify V1532 as an important residue that interacts with RAD51.     

Interactions between human BRCA2 protein and the meiosis-specific recombinase DMC1

Germline mutations in BRCA2 predispose to hereditary breast cancers. BRCA2 protein regulates recombinational repair by interaction with RAD51 via a series of degenerate BRC repeat motifs encoded by exon 11 (BRCA2(996-2113)), and an unrelated C-terminal domain (BRCA2(3265-3330)). BRCA2 is also required for meiotic recombination. Here, we show that human BRCA2 binds the meiosis-specific recombinase DMC1 and define the primary DMC1 interaction site to a 26 amino-acid region (BRCA2(2386-2411)). This region is highly conserved in BRCA2 proteins from a variety of mammalian species, but is absent in BRCA2 from Arabidopsis thaliana, Caenorhabditis elegans, and other eukaryotes. We demonstrate the critical importance of Phe2406, Pro2408, and Pro2409 at the conserved motif (2404)KVFVPPFK(2411). This interaction domain, defined as the PhePP motif, promotes specific interactions between BRCA2 and DMC1, but not with RAD51. Thus, the RAD51 and DMC1 interaction domains on BRCA2 are distinct from each other, allowing coordinated interactions of the two recombinases with BRCA2 at meiosis. These results lead us to suggest that BRCA2 is a universal regulator of RAD51/DMC1 recombinase actions.     

Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

Mammary tumors are the most common tumor type in both human and canine females. Mutations in the breast cancer susceptibility gene, BRCA2, have been found in most cases of inherited human breast cancer. Similarly, the canine BRCA2 gene locus has been associated with mammary tumors in female dogs. However, deleterious mutations in canine BRCA2 have not been reported, thus far. The BRCA2 protein is involved in homologous recombination repair via its interaction with RAD51 recombinase, an interaction mediated by 8 BRC repeats. These repeats are 26-amino acid, conserved motifs in mammalian BRCA2. Previous structural analyses of cancer-associated mutations affecting the BRC repeats have shown that the weakening of RAD51''s affinity for even 1 repeat is sufficient to increase breast cancer susceptibility. In this study, we focused on 2 previously reported canine BRCA2 mutations (T1425P and K1435R) in BRC repeat 3 (BRC3), derived from mammary tumor samples. These mutations affected the interaction of canine BRC3 with RAD51, and were considered deleterious. Two BRC3 mutations (K1440R and K1440E), reported in human breast cancer patients, occur at amino acids corresponding to those of the K1435R mutation in dogs. These mutations affected the interaction of canine BRC3 with RAD51, and may also be considered deleterious. The two BRC3 mutations and a substitution (T1430P), corresponding to T1425P in canine BRCA2, were examined for their effects on human BRC3 function and the results were compared between species. The corresponding mutations and the substitution showed similar results in both human and canine BRC3. Therefore, canine BRCA2 may be a good model for studying human breast cancer caused by BRCA2 mutations.     

High levels of BRC4 induced by a Tet-On 3G system suppress DNA repair and impair cell proliferation in vertebrate cells

Transient induction or suppression of target genes is useful to study the function of toxic or essential genes in cells. Here we apply a Tet-On 3G system to DT40 lymphoma B cell lines, validating it for three different genes. Using this tool, we then show that overexpression of the chicken BRC4 repeat of the tumor suppressor BRCA2 impairs cell proliferation and induces chromosomal breaks. Mechanistically, high levels of BRC4 suppress double strand break-induced homologous recombination, inhibit the formation of RAD51 recombination repair foci, reduce cellular resistance to DNA damaging agents and induce a G2 damage checkpoint-mediated cell-cycle arrest. The above phenotypes are mediated by BRC4 capability to bind and inhibit RAD51. The toxicity associated with BRC4 overexpression is exacerbated by chemotherapeutic agents and reversed by RAD51 overexpression, but it is neither aggravated nor suppressed by a deficit in the non-homologous end-joining pathway of double strand break repair. We further find that the endogenous BRCA2 mediates the cytotoxicity associated with BRC4 induction, thus underscoring the possibility that BRC4 or other domains of BRCA2 cooperate with ectopic BRC4 in regulating repair activities or mitotic cell division. In all, the results demonstrate the utility of the Tet-On 3G system in DT40 research and underpin a model in which BRC4 role on cell proliferation and chromosome repair arises primarily from its suppressive role on RAD51 functions.     

A variant of the breast cancer type 2 susceptibility protein (BRC) repeat is essential for the RECQL5 helicase to interact with RAD51 recombinase for genome stabilization

The BRC repeat is a structural motif in the tumor suppressor BRCA2 (breast cancer type 2 susceptibility protein), which promotes homologous recombination (HR) by regulating RAD51 recombinase activity. To date, the BRC repeat has not been observed in other proteins, so that its role in HR is inferred only in the context of BRCA2. Here, we identified a BRC repeat variant, named BRCv, in the RECQL5 helicase, which possesses anti-recombinase activity in vitro and suppresses HR and promotes cellular resistance to camptothecin-induced replication stress in vivo. RECQL5-BRCv interacted with RAD51 through two conserved motifs similar to those in the BRCA2-BRC repeat. Mutations of either motif compromised functions of RECQL5, including association with RAD51, inhibition of RAD51-mediated D-loop formation, suppression of sister chromatid exchange, and resistance to camptothecin-induced replication stress. Potential BRCvs were also found in other HR regulatory proteins, including Srs2 and Sgs1, which possess anti-recombinase activities similar to that of RECQL5. A point mutation in the predicted Srs2-BRCv disrupted the ability of the protein to bind RAD51 and to inhibit D-loop formation. Thus, BRC is a common RAD51 interaction module that can be utilized by different proteins to either promote HR, as in the case of BRCA2, or to suppress HR, as in RECQL5.     

Cancer-Associated Mutations in BRC Domains of BRCA2 Affect Homologous Recombination Induced by Rad51

The tumor suppressor BRCA2 protein plays a major role in the regulation of Rad51-catalyzed homologous recombination. BRCA2 interacts with monomeric Rad51 primarily via conserved BRC domains and coordinates the formation of Rad51 filaments at double-stranded DNA (dsDNA) breaks. A number of cancer-associated mutations in BRC4 and BRC2 domains have been reported. To elucidate their effects on homologous recombination, we studied Rad51 filament formation on single-stranded DNA and dsDNA substrates and Rad51-catalyzed strand exchange, in the presence of wild-type and mutated peptides of either BRC4 or BRC2. While the wild-type BRC2 and BRC4 peptides inhibited filament formation and, thus, strand exchange, the mutated forms decreased significantly these inhibitory effects. These results are consistent with a three-dimensional model for the interface between individual BRC repeats and Rad51. We suggest that mutations at sites crucial for the association between Rad51 and BRC domains impair the ability of BRCA2 to recruit Rad51 to dsDNA breaks, hampering recombinational repair.     

Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity

The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ～35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the analysis of protein-protein interactions for chemical biology and molecular therapeutics.     

Classical molecular dynamics simulations of the complex between the RAD51 protein and the BRC hairpin loops of the BRCA2 protein

In the repair of double-strand breaks of DNA by homologous recombination the recombinase protein RAD51 has its functions controlled by the breast cancer susceptibility protein BRCA2. BRCA2 can bind to RAD51 via the BRC repeats BRC1–BRC8, which are eight conserved sequence motifs in BRCA2 of about 35 amino acids. We have carried out a series of extensive unrestrained atomistic molecular dynamics (MD) simulations in explicit water for a total time period of 248 ns, in order to study the dynamical behaviour and conformations of the complexes between the hairpin loop region of the BRC repeats and RAD51. Our simulations have allowed us to investigate the conformations adopted by the BRC repeats both while bound to RAD51 and while isolated. These conformations are rationalised through an analysis of the inter- and intra-molecular backbone and side chain bonding interactions in all the eight human BRC repeats as well as in a single-point mutation of BRC4. The differences in sequence result in differences in the interactions between the BRC repeats and the RAD51 protein but these do not appear to disrupt the binding in any of the BRC–RAD51 complexes as there are always a number of key residues remaining which allow a sufficient number of interactions to stabilise the complexes.     

RAD51 localization and activation following DNA damage

The efficient repair of double-strand breaks in DNA is critical for the maintenance of genome stability. In response to ionizing radiation and other DNA-damaging agents, the RAD51 protein, which is essential for homologous recombination, relocalizes within the nucleus to form distinct foci that can be visualized by microscopy and are thought to represent sites where repair reactions take place. The formation of RAD51 foci in response to DNA damage is dependent upon BRCA2 and a series of proteins known as the RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), indicating that the components present within foci assemble in a carefully orchestrated and ordered manner. By contrast, RAD51 foci that form spontaneously as cells undergo DNA replication at S phase occur without the need for BRCA2 or the RAD51 paralogues. It is known that BRCA2 interacts directly with RAD51 through a series of degenerative motifs known as the BRC repeats. These interactions modulate the ability of RAD51 to bind DNA. Taken together, these observations indicate that BRCA2 plays a critical role in controlling the actions of RAD51 at both the microscopic (focus formation) and molecular (DNA binding) level.     

Design,synthesis and interaction of BRC4 analogous peptides with RAD51(241–260)

Amino Acids - Breast cancer susceptibility gene 2 (BRCA2) is an important tumor suppressor, which is participated in repair of damaged DNA by its highly conserved BRC repeat motifs regulating RAD51...     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: BRCA2: One Small Step for DNA Repair,One Giant Protein Purified

DNA damage, malfunctions in DNA repair, and genomic instability are processes that intersect at the crossroads of carcinogenesis. Underscoring the importance of DNA repair in breast and ovarian tumorigenesis is the familial inherited cancer predisposition gene BRCA2. The role of BRCA2 in DNA double-strand break repair was first revealed based on its interaction with RAD51, a central player in homologous recombination. The RAD51 protein forms a nucleoprotein filament on single-stranded DNA, invades a DNA duplex, and initiates a search for homology. Once a homologous DNA sequence is found, the DNA is used as a template for the high-fidelity repair of the DNA break. Many of the biochemical features that allow BRCA2 to choreograph the activities of RAD51 have been elucidated and include: targeting RAD51 to single-stranded DNA while inhibiting binding to dsDNA, reducing the ATPase activity of RAD51, and facilitating the displacement of the single-strand DNA binding protein, Replication Protein A. These reinforcing activities of BRCA2 culminate in the correct positioning of RAD51 onto a processed DNA double-strand break and initiate its faithful repair by homologous recombination. In this review, I will address current biochemical data concerning the BRCA2 protein and highlight unanswered questions regarding BRCA2 function in homologous recombination and cancer.