The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING domain present in both proteins. The heterodimer contains an E3 ubiquitin ligase activity and participates in multiple cellular functions such as cell cycle control, DNA repair and regulation of gene transcription, collectively aimed at maintaining genomic stability and tumor suppression. Yet, the precise role of BRCA1 E3 ligase in these cellular functions is poorly understood. We present data showing that BRCA1 ubiquitinates G2/M cell cycle proteins, cyclin B and Cdc25C, leading to their accelerated degradation via a mechanism which is independent of APC/C. BRCA1-dependent degradation of cyclin B and Cdc25C is reversed by proteasome inhibitors and is enhanced following DNA damage, which may represent a possible mechanism to prevent cyclin B and Cdc25C accumulation, a requirement for mitotic entry. Our data provides mechanistic insight into how BRCA1 E3 ligase activity regulates the G2/M cell cycle checkpoint and thus, contributes to maintenance of genomic stability.
BRCA1; cyclin B; Cdc25; ubiquitination; proteasome; G2/M cell cycle checkpoint
Mutations affecting the BRCT domains of the breast cancer–associated tumor suppressor BRCA1 disrupt the recruitment of this protein to DNA double-strand breaks (DSBs). The molecular structures at DSBs recognized by BRCA1 are presently unknown. We report the interaction of the BRCA1 BRCT domain with RAP80, a ubiquitin-binding protein. RAP80 targets a complex containing the BRCA1-BARD1 (BRCA1-associated ring domain protein 1) E3 ligase and the deubiquitinating enzyme (DUB) BRCC36 to MDC1-γH2AX–dependent lysine6- and lysine63-linked ubiquitin polymers at DSBs. These events are required for cell cycle checkpoint and repair responses to ionizing radiation, implicating ubiquitin chain recognition and turnover in the BRCA1-mediated repair of DSBs.
Although the BRCA1 tumor suppressor has been implicated in many cellular processes, the biochemical mechanisms by which it influences these diverse pathways are poorly understood. The only known enzymatic function of BRCA1 is the E3 ubiquitin ligase activity mediated by its highly conserved RING domain. In vivo, BRCA1 associates with the BARD1 polypeptide to form a heterodimeric BRCA1/BARD1 complex that catalyzes autoubiquitination of BRCA1 and trans ubiquitination of other protein substrates. In most cases, BRCA1-dependent ubiquitination generates polyubiquitin chains bearing an unconventional K6 linkage that does not appear to target proteins for proteasomal degradation. Since ubiquitin-dependent processes are usually mediated by cellular receptors with ubiquitin-binding motifs, we screened for proteins that specifically bind autoubiquitinated BRCA1. Here we report that the UBXN1 polypeptide, which contains a ubiquitin-associated (UBA) motif, recognizes autoubiquitinated BRCA1. This occurs through a bipartite interaction in which the UBA domain of UBXN1 binds K6-linked polyubiquitin chains conjugated to BRCA1 while the C-terminal sequences of UBXN1 bind the BRCA1/BARD1 heterodimer in a ubiquitin-independent fashion. Significantly, the E3 ligase activity of BRCA1/BARD1 is dramatically reduced in the presence of UBXN1, suggesting that UBXN1 regulates the enzymatic function of BRCA1 in a manner that is dependent on its ubiquitination status.
Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitin-ligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.
BRCT; DNA repair; peptide; radiation; RING; ubiquitylation
The basal-like breast cancer, a new category of breast cancer associated with poor prognosis and possibly unique chemosensitivity, is a current topic in the breast cancer field. Evidence from multiple sources strongly indicate that impairment of BRCA1 pathways is responsible for this phenotype, implying the importance of BRCA1 not only in familial breast cancers but also in sporadic cancers. BRCA1 acts as a hub protein that coordinates a diverse range of cellular pathways to maintain genomic stability. BRCA1 participates in multiple cellular supercomplexes to execute its tasks and, in most of the complexes, BRCA1 exists as a RING heterodimer with BARD1 to provide ubiquitin E3 ligase activity that is required for its tumor suppressor function. It was revealed recently that the BRCA1 RING finger is capable of catalyzing multiple types of ubiquitination depending upon the interacting E2, the ubiquitin carrier protein. BRCA1 may catalyze distinct ubiquitination on different substrates as the situation demands. On the other hand, in response to DNA double-strand breaks where BRCA1 plays its major role for homologous recombination repair, recent evidence showed that ubiquitination is a critical step to recruit BRCA1 to the damaged site through UIM (ubiquitin interacting motif) containing protein RAP80. Thus, ubiquitin and BRCA1 likely affect each other in many ways to perform cellular functions. Elucidation of this mechanism in relation to cell survival is now much anticipated because it could be a key to predict chemosensitivity of basal-like breast cancer.
Inherited mutations in the BRCA1 gene predispose to a higher risk of breast/ovarian cancer. The BRCA1 tumor suppressor is a 1863 amino acid protein with multiple protein interaction domains that facilitate its roles in regulating DNA repair and maintenance, cell cycle progression, transcription, and cell survival/apoptosis. BRCA1 was first identified as a nuclear phosphoprotein, but has since been shown to contain different transport sequences including nuclear export and nuclear localization signals that enable it to shuttle between specific sites within the nucleus and cytoplasm, including DNA repair foci, centrosomes, and mitochondria. BRCA1 nuclear transport and ubiquitin E3 ligase enzymatic activity are tightly regulated by the BRCA1 dimeric binding partner BARD1 and further modulated by cancer mutations and diverse signaling pathways. This paper will focus on the transport, dynamics, and multiple intracellular destinations of BRCA1 with emphasis on how regulation of these events has impact on, and determines, a broad range of important cellular functions.
Human BRCA1 (BRreast CAncer susceptible gene1) is known to involve in cell cycle control, transcriptional regulation, DNA recombination, DNA repair and many other processes. hBARD1 (BRCA1-Associated Ring Domain 1) forms heterodimer via its N-terminal conserved RING domain with BRCA1. In Arabidopsis, two genes, At4g21070 and At1g04020, that share N-terminal RING domain and C-terminal BRCT (for BRCA1 C-Terminal) domains with no substantial similarities for other motifs, have been identified. AtBRCA1 was induced by γ-ray while AtBARD1 was required for DNA repair. Recently, we find that AtBARD1 may function to confine WUS transcription in the shoot apical meristem organization center, together with the ATPase-dependent chromatin remodeling factor, SYD. In bard1–3 Arabidopsis knockout mutant, WUS was released to the outer layers and expressed at extremely high level comparing to wild-type. Our data suggest that BARD1 mainly function as a REPRESSOR OF WUSCHEL1 (ROW1). Extensive motif analyses carried out here showed that ROW1 possesses substantial sequence identity with a reported transcription repressor, MLL and also a potential PHD domain which recognizes histone tail codes, in its uncharacterized middle region. We suggest that ROW1 represses transcription in a chromatin-related mechanism.
Arabidopsis; BARD1; ROW1; shoot apical meristem; chromatin remodeling
Mutation of the BRCA1 tumor suppressor gene predisposes women to hereditary breast and ovarian cancers. BRCA1 forms a heterodimer with BARD1. The BRCA1/BARD1 heterodimer has ubiquitin ligase activity, considered to play crucial roles in tumor suppression and DNA damage response. Nevertheless, relevant BRCA1 substrates are poorly defined. We have developed a new approach to systematically identify the substrates of ubiquitin ligases by identifying proteins that display enhanced incorporation of His-tagged ubiquitin upon ligase co-expression; using this method, we identified several candidate substrates for BRCA1. These include scaffold attachment factor B2 (SAFB2), Tel2, as well as BARD1. BRCA1 was found to enhance SAFB protein expression and induce Tel2 nuclear translocation. Identification of the ubiquitination substrates has been a major obstacle to understanding the functions of ubiquitin ligases. The quantitative proteomics approach we devised for the identification of BRCA1 substrates will facilitate the identification of ubiquitin ligase-substrate pairs.
BRCA1; BARD1; ubiquitination; substrate; quantitative proteomics
The BRCA1 tumor suppressor has been implicated in the maintenance of chromosomal stability through homology-directed repair of DNA double-strand breaks. Much of the BRCA1 in cells forms a heterodimeric complex with a structurally related protein BARD1. We report that expression of truncated mouse or human BARD1 peptides capable of interacting with Brca1 results in a homologous-repair deficiency. Repair is mildly reduced in Brca1 wild-type cells and severely reduced in cells that harbor a Brca1 splice product deleted for exon 11. Nuclear localization of the Brca1 or BARD1 peptides is not compromised, implying that the repair deficiency is caused by a more direct effect on repair. The tumor suppressor activity of BRCA1 may require the participation of BARD1 to maintain chromosome integrity through the homologous-repair pathway.
BRCA1, a multi-domain protein, is mutated in a large percentage of hereditary breast and ovarian cancers. BRCA1 is most often mutated in three domains or regions: the N-terminal RING domain, exons 11–13, and the BRCT domain. The BRCA1 RING domain is responsible for the E3 ubiquitin ligase activity of BRCA1 and mediates interactions between BRCA1 and other proteins. BRCA1 ubiquitinates several proteins with various functions. The BRCA1 BRCT domain binds to phosphoproteins with specific sequences recognized by both BRCA1 and ATM/ATR kinases. Structural studies of the RING and BRCT domains have revealed the molecular basis by which cancer causing mutations impact the functions of BRCA1. While no structural data is available for the amino acids encoded by exons 11–13, multiple binding sites and functional domains exist in this region. Many mutations in exons 11–13 have deleterious effects on the function of these domains. In this mini-review, we examine the structure-function relationships of the BRCA1 protein and the relevance to cancer progression.
BRCA1 is a well-established tumor suppressor gene, which is frequently mutated in familial breast and ovarian cancers. The gene product of BRCA1 functions in a number of cellular pathways that maintain genomic stability, including DNA damage-induced cell cycle checkpoint activation, DNA damage repair, protein ubiquitination, chromatin remodeling, as well as transcriptional regulation and apoptosis. In this review, we discuss recent advances regarding our understanding of the role of BRCA1 in tumor suppression and DNA damage response, including DNA damage-induced cell cycle checkpoint activation and DNA damage repair.
BRCA1; DNA damage response; tumorigenesis
Germline mutations of the BRCA1 tumor suppressor gene are a major cause of familial breast and ovarian cancer. BRCA1 plays critical roles in the DNA damage response that regulates activities of multiple repair and checkpoint pathways for maintaining genome stability. The BRCT domains of BRCA1 constitute a phospho-peptide binding domain recognizing a phospho-SPxF motif (S, serine; P, proline; × varies; F, phenylalanine). The BRCT domains are frequently targeted by clinically important mutations and most of these mutations disrupt the binding surface of the BRCT domains to phosphorylated peptides. The BRCT domain and its capability to bind phosphorylated protein is required for the tumor suppressor function of BRCA1. Through its BRCT phospho-binding ability BRCA1 forms at least three mutually exclusive complexes by binding to phosphorylated proteins Abraxas, Bach1 and CTIP. The A, B and C complexes, at lease partially undertake BRCA1's role in mechanisms of cell cycle checkpoint and DNA repair that maintain genome stability, thus may play important roles in BRCA1's tumor suppressor function.
AIM: To investigate the role of [breast and ovarian cancer susceptibility 1 (BRCA1)-associated RING domain 1 (BARD1)]/BRCA1 E3-ubiquitin ligase complex in governing the stability of mutant liver X receptor-α (LXR-α) protein in coronary heart disease (CHD) subjects.
METHODS: The expression analysis of various genes was carried out by quantitative real time polymerase chain reaction and western blotting within blood mononuclear cells of human CHD subjects at various stages of coronary occlusion and their corresponding normal healthy counterparts. Immunoprecipitation experiments were performed to establish protein interactions between LXR-α and BARD1. Peripheral blood mononuclear cells were cultured and exposed to Vitamin D3 and Cisplatin to validate the degradation of mutant LXR-α protein in CHD subjects by BARD1/BRCA1 complex.
RESULTS: The expression of mutant LXR-α protein in CHD subjects was found to decrease gradually with the severity of coronary occlusion exhibiting a strong negative correlation, r = -0.975 at P < 0.001. Further, the expression of BARD1 and BRCA1 also increased with the disease severity, r = 0.895 and 0.873 respectively (P < 0.001). Immunoprecipitation studies established that BARD1/BRCA1 complex degrades mutant LXR-α via ubiquitination. The absence of functional LXR-α protein resulted in increased expression of inflammatory cytokines such as interleukin (IL)-6, IL-8 and interferon-γ and decreased expression of ABCA1 (ATP-binding cassette A1) (r = 0.932, 0.949, 0.918 and -0.902 with respect to Gensini score; P < 0.001). Additionally, cell culture experiments proved that Vitamin D3 could prevent the degradation of mutant LXR-α and restore its functional activity to some extent.
CONCLUSION: Mutant LXR-α protein in CHD subjects is degraded by BARD1/BRCA1 complex and Vitamin D3 can rescue and restore its function.
Mutant liver X receptor-α; Ubiquitination; Breast and ovarian cancer susceptibility 1-associated RING domain 1/breast and ovarian cancer susceptibility 1; Mononuclear Cells; Coronary heart disease subjects; Vitamin D3
The BRCT repeats of BRCA1 are essential for tumor suppression. Phospho-peptide affinity proteomic analysis identified a novel protein, Abraxas, that directly binds the BRCA1 BRCT repeats through a phospho-SXXF motif. Abraxas binds BRCA1 mutually exclusively with BACH1 and CTIP, forming a third Brca1 complex. Abraxas recruits the ubiquitin-interacting motif (UIM) containing protein Rap80 to BRCA1. Both Abraxas and Rap80 are required for DNA damage resistance, G2/M checkpoint control and DNA repair. Rap80 is required for a subset of Brca1-foci formation in response to IR and the UIM domains alone are capable of foci formation. The Rap80/Abraxas complex may help recruit Brca1 to DNA damage sites in part through recognition of ubiquitinated proteins.
Germline mutations of the breast cancer associated gene 1 (BRCA1) predispose women to breast and ovarian cancers. BRCA1 is a large protein with multiple functional domains and interacts with numerous proteins that are involved in many important biological processes/pathways. Mounting evidence indicates that BRCA1 is involved in all phases of the cell cycle and regulates orderly events during cell cycle progression. BRCA1 deficiency, consequently causes abnormalities in the S-phase checkpoint, the G2/M checkpoint, the spindle checkpoint and centrosome duplication. The genetic instability caused by BRCA1 deficiency, however, also triggers cellular responses to DNA damage that blocks cell proliferation and induces apoptosis. Thus BRCA1 mutant cells cannot develop further into full-grown tumors unless this cellular defense is broken. Functional analysis of BRCA1 in cell cycle checkpoints, genome integrity, DNA damage response (DDR) and tumor evolution should benefit our understanding of the mechanisms underlying BRCA1 associated tumorigenesis, as well as the development of therapeutic approaches for this lethal disease.
Germ-line mutations in BRCA1 predispose to breast and ovarian cancer. BRCA1-mutated tumors show genomic instability, mainly as a consequence of impaired recombinatorial DNA repair. Here we identify 53BP1 as an essential factor for sustaining the growth arrest induced by Brca1 deletion. Depletion of 53BP1 abrogates the ATM-dependent checkpoint response and G2 cell cycle arrest triggered by the accumulation of DNA breaks in Brca1-deleted cells. This effect of 53BP1 is specific to BRCA1 function, as 53BP1 depletion did not alleviate proliferation arrest or checkpoint responses in Brca2-deleted cells. Importantly, loss of 53BP1 partially restores the homologous recombination defect of Brca1-deleted cells and reverts their hypersensitivity to DNA-damaging agents. We find reduced 53BP1 expression in subsets of sporadic triple-negative and BRCA-associated breast cancers, indicating the potential clinical implications of our findings.
Women with mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, have an increased risk of developing breast cancer. Both BRCA1 and BRCA2 are thought to be tumour suppressor genes since the wild type alleles of these genes are lost in tumours from heterozygous carriers. Several functions have been proposed for the proteins encoded by these genes which could explain their roles in tumour suppression. Both BRCA1 and BRCA2 have been suggested to have a role in transcriptional regulation and several potential BRCA1 target genes have been identified. The nature of these genes suggests that loss of BRCA1 could lead to inappropriate proliferation, consistent with the high mitotic grade of BRCA1-associated tumours. BRCA1 and BRCA2 have also been implicated in DNA repair and regulation of centrosome number. Loss of either of these functions would be expected to lead to chromosomal instability, which is observed in BRCA1 and BRCA2-associated tumours. Taken together, these studies give an insight into the pathogenesis of BRCA-associated tumours and will inform future therapeutic strategies.
BRCA1; BRCA2; breast cancer; cell cycle checkpoints; DNA repair; gene expression
BRCA1-associated RING domain (BARD1) was identified as a protein interacting with the breast cancer gene product BRCA1. The identification of tumorigenic missense mutations within BRCA1 that impair the formation of BARD1–BRCA1 complexes, and of BARD1 mutations in breast carcinomas, sustain the view that BARD1 is involved in BRCA1-mediated tumor suppression. We have cloned the murine Bard1 gene and determined that its expression in different tissues correlates with the expression profile of Brca1. To investigate the function of Bard1, we have reduced Bard1 gene expression in TAC-2 cells, a murine mammary epithelial cell line that retains morphogenetic properties characteristic of normal breast epithelium. Partial repression of Bard1, achieved by the transfection of TAC-2 cells with plasmids constitutively expressing ribozymes or antisense RNAs, resulted in marked phenotypic changes, consisting of altered cell shape, increased cell size, high frequency of multinucleated cells, and aberrant cell cycle progression. Furthermore, Bard1-repressed cell clones overcame contact inhibition of cell proliferation when grown in monolayer cultures and lost the capacity to form luminal structures in three-dimensional collagen gels. These results demonstrate that Bard1 repression induces complex changes in mammary epithelial cell properties which are suggestive of a premalignant phenotype.
Bard1; Brca1; breast cancer; cell cycle; morphogenesis
Following genotoxic stress, the histone H2AX becomes phosphorylated at serine 139 by the ATM/ATR family of kinases. The tumor suppressor BRCA1, also phosphorylated by ATM/ATR kinases, is one of several proteins that colocalize with phospho-H2AX (γ-H2AX) at sites of active DNA repair. Both the precise mechanism and the purpose of BRCA1 recruitment to sites of DNA damage are unknown. Here we show that BRCA1 and γ-H2AX form an acid-stable biochemical complex on chromatin after DNA damage. Maximal association of BRCA1 with γ-H2AX correlates with reduced global γ-H2AX levels on chromatin late in the repair process. Since BRCA1 is known to have E3 ubiquitin ligase activity in vitro, we examined H2AX for evidence of ubiquitination. We found that H2AX is ubiquitinated at lysines 119 and 119 in vivo and that blockage of 26S proteasome function stabilizes γ-H2AX levels within cells. When BRCA1 levels were reduced, ubiquitination of H2AX was also reduced, and the cells retained higher levels of phosphorylated H2AX. These results indicate that BRCA1 is recruited into stable complexes with γ-H2AX and that the complex is involved in attenuation of the γ-H2AX repair signal after DNA damage.
The tumor suppressor BRCA1 is a nuclear shuttling protein. However, the role of BRCA1 localization in the control of its functions remains to be elucidated. Given the central role of BRCA1 in DNA damage repair, we hypothesized that depletion of nuclear BRCA1 will compromise its nuclear function in DNA repair and thereby result in enhanced cytotoxic response to DNA damage. In this study, we showed that repair of DNA double strand breaks (DSBs) required BRCA1 in the nucleus. In addition, sequestering BRCA1 in the cytosol enhanced the cytotoxic response to ionizing radiation (IR) or cisplatin in human breast and colon cancer cells. However, further genetic dissection of the mechanism of this enhanced cytotoxicity using BRCA1 mutants deficient in DSB repair unexpectedly revealed a dissociation of BRCA1’s function in DNA repair from its effects on cellular sensitivity to DNA damage. Interestingly, we observed a dependence of the DNA damage-induced cell killing on the translocation and accumulation of BRCA1 in the cytosol. Together, these data suggest a novel role of cytoplasmic translocation of BRCA1, not only in controlling its DNA repair functions, but also in the regulation of cell death processes following DNA damage. Further dissection of the mechanism of cytotoxicity induced by BRCA1 cytoplasmic translocation revealed involvement of the apoptotic pathway. We propose that the status of BRCA1 nuclear/cytoplasmic shuttling may provide a molecular marker to predict tumor response and a potential novel target to sensitize cancer cells to DNA damage-based therapy.
BRCA1; nuclear export; apoptosis; DNA repair; homologous recombination; radiation sensitization; checkpoint
BRCA1 and BRCA2 are two major breast and ovarian cancer susceptibility genes. BRCA1 was the first discovered and has been a focus of research for these cancers. BRCA1 mediates tumor suppression in part through pleiotropic interactions with a network of DNA repair proteins on chromatin. BRCA1 mutations cause homologous recombination-mediated DNA repair deficiency, genomic instability, and DNA-damaging agent hypersensitivity. Although BRCA1 and BRCA2 have some shared functions in cancer predisposition and therapy response, there are also key differences indicating divergent roles for each protein. This review summarizes and highlights recent insights into the molecular events responsible for BRCA1 tumor suppression, emphasizing the DNA repair function of BRCA1 as a nexus between its roles in cancer development and therapy.
Fanconi anemia (FA) is a rare genetic disorder characterized by aplastic anemia, cancer/leukemia susceptibility and cellular hypersensitivity to DNA crosslinking agents, such as cisplatin. To date, 12 FA gene products have been identified, which cooperate in a common DNA damage-activated signaling pathway regulating DNA repair (the FA pathway). Eight FA proteins form a nuclear complex harboring E3 ubiquitin ligase activity (the FA core complex) that, in response to DNA damage, mediates the monoubiquitylation of the FA protein FANCD2. Monoubiquitylated FANCD2 colocalizes in nuclear foci with proteins involved in DNA repair, including BRCA1, FANCD1/BRCA2, FANCN/PALB2 and RAD51. All these factors are required for cellular resistance to DNA crosslinking agents. The inactivation of the FA pathway has also been observed in a wide variety of human cancers and is implicated in the sensitivity of cancer cells to DNA crosslinking agents. Drugs that inhibit the FA pathway may be useful chemosensitizers in the treatment of cancer.
Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; ).
The breast cancer 1 and 2, early onset (BRCA1 and BRCA2) genes are important for double-strand break repair by homologous recombination. Cells with inactivating mutations of the BRCA1 or BRCA2 tumor suppressor genes show increased sensitivity to Poly-ADP ribose polymerase (PARP)-inhibitors in vitro. Sporadic breast tumors with BRCA1 promoter hypermethylation show a similar phenotype to familial BRCA1 patient tumors termed “BRCAness.” Sporadic ovarian tumors with functional inactivation of BRCA1 by hypermethylation will also have the BRCA-deficiency phenocopy. The loss of BRCA1 expression associated with promoter hypermethylation will disrupt BRCA-associated DNA repair and may sensitize tumors to BRCA-directed therapies. Thus, the determination of methylation status of BRCA1 may be an important predictive classifier of response to PARP-inhibitor therapy. The methylation, and thereby functional, status of other genes implicated in the wider BRCA/homologous recombination (HR) pathway may also be relevant to the prediction of response to PARP-inhibitor therapy. Here, we describe the four optimal technologies for assaying the promoter methylation status of BRCA1 and/or other genes.
BRCA1; Hypermethylation; PARP; Bisulfite sequencing; Pyrosequencing; Quantitative MSP; Methylation beadchip
BRCA1 is a tumor suppressor with critical roles in the maintenance of genomic stability. It encodes a large protein with an amino-terminal RING domain that possesses ubiquitin-ligase activity. Given the occurrence of numerous cancer-causing mutations within its RING domain, investigators have long suspected that BRCA1's ubiquitin ligase is important for its tumor suppression and DNA repair activities. Using genetically engineered mouse models, two recent studies shed light on this age-old hypothesis.
Human BRCA2, a breast and ovarian cancer suppressor, binds to the DNA recombinase RAD51 through eight conserved BRC repeats, motifs of ∼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 (BRCA2BRC1–8) with 1127 residues spanning all 8 BRC repeats but excluding the C-terminal DNA-binding domain (BRCA2CTD). BRCA2BRC1–8 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, BRCA2BRCI–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 BRCA2CTD through a previously unrecognized role in control of RAD51 activity.