BRCA1 modulates ionizing radiation-induced nuclear focus formation by the replication protein A p34 subunit

Mutations in BRCA1 account for a significant proportion of familial breast and ovarian cancers. BRCA1 has been implicated in DNA damage responses including double-strand break (DSB) repair. However, its exact role in DSB repair and its functional relationship with other known repair proteins remain to be elucidated. In this study, we carried out a cytological analysis of the effect of BRCA1 on damage-induced nuclear focus formation mediated by the replication protein A (RPA). RPA is a multi-functional protein that participates in both DNA replication and various types of DNA repair including DSB repair. Following ionizing radiation (IR), RPA and BRCA1 formed punctate nuclear staining patterns that co-localized with each other, consistent with the implicated roles of both proteins in the same repair process. The number of damage-induced RPA foci in BRCA1-deficient cells, however, was significantly greater than that in BRCA1-positive cells. Moreover, the effect of BRCA1 on the RPA staining pattern appeared to be specific for IR but not ultraviolet (UV) irradiation. These data suggest that BRCA1 plays an important role in processing the RPA-associated intermediates during DSB repair.     

Identification of sequences that target BRCA1 to nuclear foci following alkylative DNA damage

BRCA1 is a tumor suppressor involved in the maintenance of genome integrity. BRCA1 co-localizes with DNA repair proteins at nuclear foci in response to DNA double-strand breaks caused by ionizing radiation (IR). The response of BRCA1 to agents that elicit DNA single-strand breaks (SSB) is poorly defined. In this study, we compared chemicals that induce SSB repair and observed the most striking nuclear redistribution of BRCA1 following treatment with the alkylating agent methyl methanethiosulfonate (MMTS). In MCF-7 breast cancer cells, MMTS induced movement of endogenous BRCA1 into distinctive nuclear foci that co-stained with the SSB repair protein XRCC1, but not the DSB repair protein gamma-H2AX. XRCC1 did not accumulate in foci after ionizing radiation. Moreover, we showed by deletion mapping that different sequences target BRCA1 to nuclear foci induced by MMTS or by ionizing radiation. We identified two core MMTS-responsive sequences in BRCA1: the N-terminal BARD1-binding domain (aa1-304) and the C-terminal sequence aa1078-1312. These sequences individually are ineffective, but together they facilitated BRCA1 localization at MMTS-induced foci. Site-directed mutagenesis of two SQ/TQ motif serines (S1143A and S1280A) in the BRCA1 fusion protein reduced, but did not abolish, targeting to MMTS-inducible foci. This is the first report to describe co-localization of BRCA1 with XRCC1 at SSB repair foci. Our results indicate that BRCA1 requires BARD1 for targeting to different types of DNA lesion, and that distinct C-terminal sequences mediate selective recruitment to sites of double- or single-strand DNA damage.     

The role of the BRCA1 tumor suppressor in DNA double-strand break repair

The tumor suppressor gene BRCA1 was cloned in 1994 based on its linkage to early-onset breast and ovarian cancer. Although the BRCA1 protein has been implicated in multiple cellular functions, the precise mechanism that determines its tumor suppressor activity is not defined. Currently, the emerging picture is that BRCA1 plays an important role in maintaining genomic integrity by protecting cells from double-strand breaks (DSB) that arise during DNA replication or after DNA damage. The DSB repair pathways available in mammalian cells are homologous recombination and nonhomologous end-joining. BRCA1 function seems to be regulated by specific phosphorylations in response to DNA damage and we will focus this review on the roles played by BRCA1 in DNA repair and cell cycle checkpoints. Finally, we will explore the idea that tumor suppression by BRCA1 depends on its control of DNA DSB repair, resulting in the promotion of error-free and the inhibition of error-prone recombinational repair.     

Constitutive association of BRCA1 and c-Abl and its ATM-dependent disruption after irradiation

BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.     

The NuRD chromatin–remodeling complex regulates signaling and repair of DNA damage

Cells respond to ionizing radiation (IR)–induced DNA double-strand breaks (DSBs) by orchestrating events that coordinate cell cycle progression and DNA repair. How cells signal and repair DSBs is not yet fully understood. A genome-wide RNA interference screen in Caenorhabditis elegans identified egr-1 as a factor that protects worm cells against IR. The human homologue of egr-1, MTA2 (metastasis-associated protein 2), is a subunit of the nucleosome-remodeling and histone deacetylation (NuRD) chromatin-remodeling complex. We show that knockdown of MTA2 and CHD4 (chromodomain helicase DNA-binding protein 4), the catalytic subunit (adenosine triphosphatase [ATPase]) of NuRD, leads to accumulation of spontaneous DNA damage and increased IR sensitivity. MTA2 and CHD4 accumulate in DSB-containing chromatin tracks generated by laser microirradiation. Directly at DSBs, CHD4 stimulates RNF8/RNF168-dependent formation of ubiquitin conjugates to facilitate the accrual of RNF168 and BRCA1. Finally, we show that CHD4 promotes DSB repair and checkpoint activation in response to IR. Thus, the NuRD chromatin–remodeling complex is a novel regulator of DNA damage responses that orchestrates proper signaling and repair of DSBs.     

Structural insights into BRCA2 function

BRCA2 is a tumor suppressor directly implicated in familial breast cancer. Extensive genetic and biochemical characterization has shown that BRCA2 is involved in the maintenance of chromosomal stability and that it has an important role in recombination-mediated double-strand DNA break repair. Two recent structures of BRCA2 domains have revealed that it may serve as a critical mediator of DNA repair through direct interactions with Rad51, the eukaryotic homolog of RecA, and with single-stranded DNA. Before the structures were determined, little was known about the structural basis of BRCA2 interactions with the Rad51 pathway of DNA repair. Taken together, the structures provide striking insights into the role of BRCA2 in double-strand DNA break repair and suggest a direct role for BRCA2 in homologous recombination that was not evident from earlier studies.     

Recognition of DNA double strand breaks by the BRCA1 tumor suppressor network

DNA double-strand breaks (DSBs) occur in response to both endogenous and exogenous genotoxic stress. Inappropriate repair of DSBs can lead to either loss of viability or to chromosomal alterations that increase the likelihood of cancer development. In strong support of this assertion, many cancer predisposition syndromes stem from germline mutations in genes involved in DNA DSB repair. Among the most prominent of such tumor suppressor genes are the Breast Cancer 1 and Breast Cancer 2 genes (BRCA1 and BRCA2), which are mutated in familial forms of breast and ovarian cancer. Recent findings implicate BRCA1 as a central component of several distinct macromolecular protein complexes, each dedicated to distinct elements of DNA DSB repair and tumor suppression. Emerging evidence has shed light on some of the molecular recognition processes that are responsible for targeting BRCA1 and its associated partners to DNA and chromatin directly flanking DSBs. These events are required for BRCA1-dependent DNA repair and tumor suppression. Thus, a detailed temporal and spatial knowledge of how breaks are recognized and repaired has profound implications for understanding processes related to the genesis of malignancy and to its treatment.     

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate–dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21^Cip1 accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.     

BRCA1 Pathway Function in Basal-Like Breast Cancer Cells

Sporadic basal-like cancers (BLCs) are a common subtype of breast cancer that share multiple biological properties with BRCA1-mutated breast tumors. Despite being BRCA1^+/+, sporadic BLCs are widely viewed as phenocopies of BRCA1-mutated breast cancers, because they are hypothesized to manifest a BRCA1 functional defect or breakdown of a pathway(s) in which BRCA1 plays a major role. The role of BRCA1 in the repair of double-strand DNA breaks by homologous recombination (HR) is its best understood function and the function most often implicated in BRCA1 breast cancer suppression. Therefore, it is suspected that sporadic BLCs exhibit a defect in HR. To test this hypothesis, multiple DNA damage repair assays focused on several types of repair were performed on a group of cell lines classified as sporadic BLCs and on controls. The sporadic BLC cell lines failed to exhibit an overt HR defect. Rather, they exhibited defects in the repair of stalled replication forks, another BRCA1 function. These results provide insight into why clinical trials of poly(ADP-ribose) polymerase (PARP) inhibitors, which require an HR defect for efficacy, have been unsuccessful in sporadic BLCs, unlike cisplatin, which elicits DNA damage that requires stalled fork repair and has shown efficacy in sporadic BLCs.     

A conserved pathway to activate BRCA1-dependent ubiquitylation at DNA damage sites

The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We show here that the Caenorhabditis elegans BRCA1/BARD1 (CeBCD) complex possesses an E3-Ub ligase responsible for ubiquitylation at DNA damage sites following ionizing radiation (IR). The DNA damage checkpoint promotes the association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), leading to the formation of an active E3-Ub ligase on chromatin following IR. Correspondingly, defects in Ubc5(let-70) or the DNA damage checkpoint genes atl-1 or mre-11 abolish CeBCD-dependent ubiquitylation in vivo. Extending these findings to human cells reveals a requirement for UbcH5c, the MRN complex, gamma-H2AX and a co-dependence for ATM and ATR kinases for BRCA1-dependent ubiquitylation at DNA damage sites. Furthermore, we demonstrate that the DNA damage checkpoint promotes the association between BRCA1 and UbcH5c to form an active E3-Ub ligase on chromatin after IR. These data reveal that BRCA1-dependent ubiquitylation is activated at sites of DNA repair by the checkpoint as part of a conserved DNA damage response.     

Cell cycle-dependent complex formation of BRCA1.CtIP.MRN is important for DNA double-strand break repair

BRCA1 plays an important role in the homologous recombination (HR)-mediated DNA double-strand break (DSB) repair, but the mechanism is not clear. Here we describe that BRCA1 forms a complex with CtIP and MRN (Mre11/Rad50/Nbs1) in a cell cycle-dependent manner. Significantly, the complex formation, especially the ionizing radiation-enhanced association of BRCA1 with MRN, requires cyclin-dependent kinase activity. CtIP directly interacts with Nbs1. The in vivo association of BRCA1 with MRN is largely dependent on the association of CtIP with the BRCT domains at the C terminus of BRCA1, whereas the N terminus of BRCA1 also contributes to its association with MRN. CtIP, as well as the interaction of BRCA1 with CtIP and MRN, is critical for IR-induced single-stranded DNA formation and cellular resistance to radiation. Consistently, CtIP itself is required for efficient HR-mediated DSB repair, like BRCA1 and MRN. These studies suggest that the complex formation of BRCA1.CtIP.MRN is important for facilitating DSB resection to generate single-stranded DNA that is needed for HR-mediated DSB repair. Because cyclin-dependent kinase is important for establishing IR-enhanced interaction of MRN with BRCA1, we propose that the cell cycle-dependent complex formation of BRCA1, CtIP, and MRN contributes to the activation of HR-mediated DSB repair in the S and G(2) phases of the cell cycle.     

BRCA2 is required for homology-directed repair of chromosomal breaks

The BRCA2 tumor suppressor has been implicated in the maintenance of chromosomal stability through a function in DNA repair. In this report, we examine human and mouse cell lines containing different BRCA2 mutations for their ability to repair chromosomal breaks by homologous recombination. Using the I-SceI endonuclease to introduce a double-strand break at a specific chromosomal locus, we find that BRCA2 mutant cell lines are recombination deficient, such that homology-directed repair is reduced 6- to >100-fold, depending on the cell line. Thus, BRCA2 is essential for efficient homology-directed repair, presumably in conjunction with the Rad51 recombinase. We propose that impaired homology-directed repair caused by BRCA2 deficiency leads to chromosomal instability and, possibly, tumorigenesis, through lack of repair or misrepair of DNA damage.     

ATM activation by ionizing radiation requires BRCA1-associated BAAT1

ATM (ataxia telangiectasia mutated) is required for the early response to DNA-damaging agents such as ionizing radiation (IR) that induce DNA double-strand breaks. Cells deficient in ATM are extremely sensitive to IR. It has been shown that IR induces immediate phosphorylation of ATM at Ser(1981), leading to catalytic activation of the protein. We recently isolated a novel BRCA1-associated protein, BAAT1 (BRCA1-associated protein required for ATM activation-1), by yeast two-hybrid screening and found that BAAT1 also binds to ATM, localizes to double-strand breaks, and is required for Ser(1981) phosphorylation of ATM. Small interfering RNA-mediated stable or transient reduction of BAAT1 resulted in decreased phosphorylation of both ATM at Ser(1981) and CHK2 at Thr(68). Treatment of BAAT1-depleted cells with okadaic acid greatly restored phosphorylation of ATM at Ser(1981), suggesting that BAAT1 is involved in the regulation of ATM phosphatase. Protein phosphatase 2A-mediated dephosphorylation of ATM was partially blocked by purified BAAT1 in vitro. Significantly, acute loss of BAAT1 resulted in increased p53, leading to apoptosis. These results demonstrate that DNA damage-induced ATM activation requires a coordinated assembly of BRCA1, BAAT1, and ATM.     

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.     

DNA repair deficiency as a therapeutic target in cancer

Inhibitors of DNA repair proteins have been used in cancer therapy, mostly to potentiate the effects of cytotoxic agents. However, tumor cells frequently exhibit deficiencies in the signalling or repair of DNA damage. These deficiencies probably contribute to pathogenesis of the disease, but they also present an opportunity to target the tumor. Recently, inhibitors of poly(ADP-ribose) polymerase (PARP) have been shown to be highly selective for tumor cells with defects in the repair of double-strand DNA breaks (DSBs) by homologous recombination, particularly in the context of BRCA1 or BRCA2 mutation. It seems likely that other DNA repair processes can be targeted in a similar manner. These synthetic lethal approaches highlight how an understanding of DNA repair processes can be used in the development of novel cancer treatments.     

BRCA1 associates with the inactive X chromosome in late S-phase, coupled with transient H2AX phosphorylation

The BRCA1 tumor suppressor gene encodes an E3-ubiquitin ligase that has been implicated in several distinct biochemical processes. As the cell cycle progresses, BRCA1 proteins interact transiently with nuclear foci containing DNA replication and DNA double-strand repair machinery. A hallmark of these foci is the presence of S139 phosphorylated histone H2AX. BRCA1 was recently shown to associate with facultative heterochromatin at the inactive X chromosome (Xi), where it may play a role in maintaining gene silencing. As the kinetics of this interaction has not been described, we sought to establish whether association of BRCA1 with the Xi also correlated with replication. Here we demonstrate that the interaction of BRCA1 and the Xi is transient, occurring during late S-phase. This interaction is concomitant with the presence of distinct foci of S139 phospho-H2AX and specifically corresponds with late replication of the Xi. BRCA1 and phospho-H2AX appear on the Xi immediately adjacent to CAF-1, a known marker of replication fork activity. Taken together, these data implicate BRCA1 and the H2AX kinase in replication of facultative heterochromatin on the Xi, most likely in a fashion similar to that performed at sites of DNA replication and double-strand break repair observed on somatic chromosomes.