Investigating contributions of BRCA2 DNA-binding domains toward maintaining genome integrity

摘要 存在着消除DNA损伤的保守机制，如果不修复，可能会导致基因突变和 基因组重排触发肿瘤细胞转化和肿瘤发生。特别是，同源的 重组(HR)是修复DNA双链断裂(DSB)、链间断裂的高保真工具 交联链和折叠的DNA复制叉子。HR的正确执行需要肿瘤抑制基因BRCA2 (乳腺癌2)。BRCA2的种系突变导致乳腺癌和卵巢癌以及癌症的易感性 范可尼贫血综合征。此外，在各种癌症类型中都发现了体细胞驱动基因BRCA2突变。 BRCA2在HR修复中的作用机制有待进一步研究。重要的是，这项研究 在此提出的将有助于确定两个不同的DNA结合域对功能的贡献 BRCA2作为“重组介体”，特别是在促进RAD51的复合体的组装方面 双链双链DNA加工过程中的重组酶。我们的中心假设是 BRCA2中的两个DNA结合域在指导BRCA2依赖的DSB修复和 通过参与单链DNA和DNA结构的复制叉保存 已处理的DSB。我们将在两个具体的项目下进行各种生化、遗传和细胞生物学研究 旨在检验这一核心信条。通过这些实验，我们将描绘出基因突变是如何在 每个DNA结合域都影响HR和复制分叉保存的关键方面。由此产生的结果是 联谊项目不仅将扩大我们对BRCA2DNA结合域如何有助于 维持基因组的稳定性，但它们也将阐明这些区域内的突变是如何 最终导致恶变。此外，我们希望我们的发现有助于确定新的靶点和途径支点 为新的癌症治疗方法的发展指明了方向。

ABSTRACT Conserved mechanisms exist to eliminate DNA damage, which, if left unrepaired, can cause gene mutations and genome rearrangements to trigger neoplastic cell transformation and oncogenesis. In particular, homologous recombination (HR) represents a high-fidelity tool for the repair of DNA double-strand breaks (DSBs), interstrand crosslinks, and collapsed DNA replication forks. Proper execution of HR requires the tumor suppressor BRCA2 (Breast Cancer 2). Germline mutations in BRCA2 lead to breast and ovarian cancer and also the cancer-prone syndrome Fanconi anemia. Moreover, somatic driver BRCA2 mutations are found in a variety of cancer types. Further work is necessary to elucidate the mechanistic role of BRCA2 in HR repair. Importantly, the research proposed herein will help define the contributions of two distinct DNA binding domains toward the function of BRCA2 as a “recombination mediator,” specifically in facilitating the assembly of complexes of the RAD51 recombinase on single-stranded DNA derived from the processing of DSBs. Our central hypothesis posits that the two DNA-binding domains within BRCA2 play an essential role in guiding BRCA2-dependent DSB repair and replication fork preservation through the engagement of single-stranded DNA and DNA structures present in processed DSBs. We will conduct a variety of biochemical, genetic, and cell biological studies under two specific aims to test this central tenet. Throughout these experiments, we will delineate how genetic mutations within each DNA binding domain affect key aspects of HR and replication fork preservation. The results from this fellowship project will not only expand our understanding of how the BRCA2 DNA-binding domains contribute to the maintenance of genome stability, but they will also shed light on how mutations within these domains ultimately cause malignancy. Furthermore, we expect our findings to help identify new targets and pathway pivot points for the development of novel cancer therapeutics.