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    Awarded Grants
    Role of the BRCA1-FANCD2 Pathway in the Repair of DNA Double-Strand Breaks by Non-Homologous Recombination

    Scientific Abstract:
    This basic research proposal seeks to characterize the role of BRCA1 and FANCD2 in the repair of DNA double-strand breaks (DSBs) by non-homologous recombination (NHR). BRCA1 is mutated in a substantial proportion of inherited breast cancers and accumulating evidence suggests that inactivation of various pathways controlled by BRCA1 through genetic or epigenetic modification may also contribute to sporadic breast cancer development. BRCA1 functionally interacts with the Fanconi Anemia (FA) gene product FANCD2, but the molecular mechanisms by which the BRCA1-FANCD2 pathway protects against genomic instability and carcinogenesis remain to be fully characterized. DSBs are a deleterious form of DNA damage that can lead to cell death or malignant cell transformation. BRCA1 and more recently also the FA genes have been implicated in the regulation of DSB repair by homologous recombination (HR). However, the function of these genes in the regulation of the other principal repair pathway, NHR, which is thought to be the dominant repair pathway in human cells, is poorly characterized. This study will employ genetic analysis to clarify the roles of BRCA1 and FANCD2 in NHR. We will utilize novel recombination substrates to detect the repair of site-specific DSBs in living cells. Three different break types will be generated at recognition sites within the chromosomally integrated plasmid substrates. We will determine the proficiency and the fidelity of NHR repair in a panel of isogenic human cell pairs (breast carcinoma cells and normal fibroblasts). Our main working hypothesis is that the BRCA1-FANCD2 pathway controls NHR processes to limit the genetic changes associated with DSB repair. The Specific Aims are to determine (1) the role of BRCA1 in the repair of site-specific DSBs via NHR, (2) the role of FANCD2 in NHR, and (3) the role of the BRCA1-FANCD2 interaction in NHR as well as in the removal of breaks caused by certain chemotherapeutic agents (such as bleomycin or etoposide), which also requires NHR. Defective recombination in breast cancer cells is a more widespread cause of genomic instability than previously appreciated. Conversely, such cells may be more sensitive to certain drug-induced DNA breaks which are repaired by NHR (and/or HR). It is therefore important that recombinational processes are understood fully at the molecular level.

    Lay Abstract:
    Breast cancer is the most common type of cancer among American women, affecting about 1 in 8 during their lifetime. About 5-10% of breast cancers are inherited. The Breast Cancer gene 1 (BRCA1) is mutated in a substantial proportion of inherited breast cancers. We know that BRCA1 plays an important role in the repair of broken chromosomes. A chromosomal break is caused by a disruption of the DNA molecule that forms the chromosome. Since a DNA molecule consists of two strands, such a break is termed “double-strand break” (DSB). DSBs may be produced by external factors, such as X-rays or chemotherapeutic agents used in cancer therapy. DSBs pose a major threat to the genetic integrity of a cell. If not properly repaired, a DSB can lead to cell death or to genetic changes which in turn can result in cancer development. BRCA1 is known to promote DSB repair via a pathway called homologous recombination, but its role in the other major repair pathway, non-homologous recombination (NHR), is poorly understood. Considerable interest has been generated by recent discoveries that link the function of BRCA1 to a rare childhood disorder called Fanconi Anemia (FA). BRCA1 functionally interacts with the FA gene product D2 (FANCD2), but it not known how this interaction protects against chromosomal breakage and breast cancer development. This research proposal seeks to improve our understanding of the role of BRCA1 and FANCD2 in the repair of DSBs by NHR. Our study will utilize novel plasmids, which are artificial DNA molecules, containing test genes. NHR will be measured within these test genes. These plasmids will be introduced into human cells and inserted into a chromosomal location. These cells have been obtained from individuals suffering from breast cancer or FA and have been grown in a nutrient medium in-vitro. We will study the efficiency and the quality of the repair of DSBs produced within the test genes. We will then change the genetic status of the cells, for example by adding back BRCA1 into BRCA1-deficient cells, and observe how this improves DSB repair. We believe that defects in DSB repair, which promote breast cancer development by compromising genetic integrity, may also represent the Achilles' Heel of the cancers arising in this setting by offering a molecular target for novel cancer therapies.