Discovery of the BRCA1 gene and the direct correlation with high rates of breast and ovarian cancer progression has contributed greatly to our understanding of tumor suppressor functions and their role in cancer development. The BRCA1 gene was first discovered using positional cloning methods on genomic sequences from patients with familial, hereditary breast and ovarian cancers. The BRCA1 gene is located on the long arm of chromosome 17 at band 21. There are hundreds of different mutations shown to occur in the BRCA1 gene, although they do not always manifest as disease, for example in cases where there is only partial loss of protein functions. The most deleterious mutations in BRCA1 are frameshift and nonsense mutations that result in early stop codons, causing the protein to be degraded or severely truncated. Much of the characterization of BRCA1 mutations is based on studies done with patients that have haploinsufficiency in the BRCA1 gene, having only one wild-type copy of the gene in diploid cells. In mouse models, the absence of at least one wild-type copy of the BRCA1 gene is an embryonic lethal mutation, indicating a critical role for BRCA1 during development. In addition, the absence of homologous genes in most non-mammalian model systems has made the characterization of BRCA1 more difficult.
Mutations in the BRCA1 gene thus far have only been correlated with the development of breast and ovarian cancers, although ongoing studies suggest there may be another small subset of pancreatic and prostate cancers that can result from BRCA1 haploinsufficiency. The loss of heterozygosity or sporadic loss of both BRCA1 alleles is not uncommon in many types of cancers, including sporadic breast cancer. The role of BRCA1 in non-specific sporadic tumor formation is still unclear, but the characterization of BRCA1 mutations in patient tumors has allowed clinicians to optimize treatment regimens for a subset of cancer patients and the status of BRCA1 protein ex
The protein product of the BRCA1 gene is a large protein (~220 kD) that has two BRCT domains, a RING domain, and a DNA-binding domain. The BRCT domains of BRCA1 function as phospho-protein binding domains that mediate a large number of protein interactions by BRCA1. The proteins that BRCA1 interact with are involved with DNA repair, chromatin remodeling, cell cycle control, transcriptional regulation, ubiquitin ligation and apoptosis. The large number of interactions with DNA repair and chromatin remodeling factors has led to characterization of the BRCA1-associated signaling complex (BASC), an aggregation of proteins that function to sense DNA damage, alter DNA topology at damage sites, and recruit repair factors to the sites of damage. The DNA-binding domain of BRCA1 binds to DNA molecules of >300-500 bp, and has a higher affinity for complex DNA structures such as cruciform DNA rather than single-stranded DNA or linear double-stranded DNA. Interestingly, BRCA1 formed looped structures when incubated with linear DNA, and showed the highest affinity for plasmid DNA, indicating a circular structural preference. DNA damage detection and signaling, and the choice of repair pathways also depends on BRCA1 and its binding partners. The BRCA1 protein often coprecipitates in vivo and in vitro with BARD1 that has ubiquitin ligase functions, and CtIP, a nuclease that resects DNA ends following a DSB. Many of the functions of BRCA1 depend on its association with other proteins such as BARD1 and BACH1, and disruption of these interactions is thought to promote tumor development. To date, elucidating molecular functions of the BRCA1 protein has shed light on several possible mechanisms of tumor origination, including suppression of estrogen-mediated transcription, induction of apoptosis, mitotic spindle formation, and DNA repair. The focus of our investigation is centered on the mechanisms of BRCA1 inhibition of nuclease-mediated DSB resection activities.
Throughout the cell cycle, BRCA1 undergoes several phosphorylation events to regulate its protein interaction activities during the DNA damage response. Following DNA damage, BRCA1 is phosphorylated by ataxia telangiectasia mutated (ATM), and ataxia telangiectasia-related (ATR) kinases in response to DSBs or DNA adducts, respectively. Additionally, the phosphorylation of BRCA1 by checkpoint homolog 2 (Chk2) can regulate the activities of BRCA1 to prevent chromosome instability and control DSB repair pathway choices. The phosphorylation of BRCA1 following DNA damage is functionally redundant to ensure its proper regulation, an indication of the central role BRCA1 plays in the cellular responses to DNA damage. The interaction of BRCA1 with Cterminus interacting protein (CtIP) has been shown to regulate the choice of DSB repair pathway, and this interaction is also implicated in the resection of DNA ends following a DSB. Additionally, BRCA1 ubiquitinates CtIP in a phosphorylation-dependent manner, and together they are implicated in the removal of covalent modifications on the ends of a DSB. BRCA1 also interacts with the Mre11, Rad50, Nbs1 (MRN) complex at the sites of DNA damage following phosphorylation, and inhibits the exonuclease activity of Mre11 in vitro by binding directly to DNA. In order to clarify the role of BRCA1 in DSB mutagenesis, we will test the hypothesis that BRCA1 inhibits Mre11 from mutagenic nuclease resection activities during MMEJ and NHEJ.
The generation of a DSB may be due to normal cellular processes, such as estrogen metabolism, or can be as a result of exogenous and endogenous mutagens. Many exogenous sources such as radiation and pollution are responsible for causing DSBs, but the occurrence of a DSB can also be useful during meiosis and the generation of genetic diversity in lymphocytic tissues. Normal cellular activities often result in DSBs, such as during replication in mitotic cells or transcription. However, damage-induced DSBs are considered the most detrimental genomic insult a cell can sustain, as even a single unrepaired break can lead to cell death or malignancy.
Two major distinct pathways of double-strand break (DSB) repair are thought to function in mammalian cells: homologous recombination (HR) and non-homologous end joining. Efficient repair of a DSB by HR is known to depend upon BRCA1 protein function and interactions with Rad51 and replication protein A (RPA). Numerous in vivo and in vitro studies also suggest that BRCA1 influences the frequency and fidelity of NHEJ repair of DSBs during all phases of the cell cycle, including when homologous recombination is active. However, one question that remains unanswered is what role BRCA1 plays in NHEJ and other “alternative” end joining repair pathways.