Project 1: Recruitment of the BRCA1-associated Homologous Recombination Machinery

项目1：BRCA1相关同源重组机制的招募

基本信息

批准号：
10396607
负责人：
Samuel Bunting
金额：
$ 38.43万
依托单位：
RBHS -CANCER INSTITUTE OF NEW JERSEY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10396607
关键词：
Address Affect Aging Appearance Automobile Driving BARD1 gene BRCA1 Mutation BRCA1 gene BRCA2 gene Breast Cancer Cell Breast Cancer Model Breast Cancer Patient Cancer Model Cell Line Cells Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Complex DNA DNA Damage DNA Double Strand Break DNA Repair DNA Repair Disorder DNA Repair Gene DNA biosynthesis DNA replication fork Data Double Strand Break Repair Event Goals Hereditary Breast and Ovarian Cancer Syndrome Incidence Investigation Knock-out Knockout Mice Literature Malignant Neoplasms Malignant neoplasm of ovary Malignant neoplasm of pancreas Mass Spectrum Analysis Measures Mediating Mediator of activation protein Methods Microscopy Modeling Mus Mutation PALB2 gene Pathologic Pathway interactions Pattern Phenotype Phosphorylation Play Poly(ADP-ribose) Polymerases Post-Translational Protein Processing Predisposition Prevention Protein Isoforms Protein Methylation Protein Methyltransferases Proteins Proteomics Regulation Reporting Research Role Scaffolding Protein Signal Transduction Site Somatic Mutation Sumoylation Pathway TOPBP1 Gene TP53 gene Tertiary Protein Structure Testing Time Tumor Suppressor Proteins Ubiquitin Ubiquitination Work base cancer type cell growth cohort conditional knockout design effective therapy homologous recombination inhibitor malignant breast neoplasm medulloblastoma mouse model mutant mutant mouse model novel novel strategies p53-binding protein 1 prevent protein complex recruit repaired replication stress response treatment response tumor tumorigenesis ubiquitin-protein ligase

项目摘要

PROJECT SUMMARY: “RECRUITMENT OF THE BRCA1-ASSOCIATED HOMOLOGOUS RECOMBINATION MACHINERY” A substantial proportion of tumors show patterns of mutation that arise because of problems with DNA repair. Deficiencies in key DNA repair genes, such as BRCA1, PALB2 and BRCA2, are associated with inefficient DNA repair by homologous recombination (HR). Each of these factors is required to load the key HR intermediate, RAD51, at DNA double-strand break sites. The exact mechanism by which BRCA1 is recruited to DNA break sites and becomes activated is not fully understood. Efforts to design effective therapies to counteract DNA repair deficiencies are undermined by this lack of understanding of BRCA1 function. The overall goal of this proposal is to produce an integrated model for how BRCA1 becomes localized and activated at DNA break sites. This goal will be realized by investigating three mechanisms that control the recruitment of the BRCA1-associated repair machinery. First, the contribution of BARD1 for BRCA1 recruitment will be evaluated. BARD1 is a RING domain protein that partners with BRCA1, and appears to control the recruitment of BRCA1 to DNA break sites in the early time points after DNA damage. Preliminary results with novel mouse models have demonstrated that the interaction of BARD1 with BRCA1 is essential for normal DNA repair. Work in this proposal will clarify the mechanism by which BARD1 controls BRCA1 repair activities. Secondly, the mechanism of TOPBP1-dependent recruitment of BRCA1 will be identified. TOPBP1 is a large scaffold protein that loads onto chromatin at DNA damage sites and recruits multiple DNA repair factors. Through the use of advanced proteomic methods to study the protein interaction networks of BRCA1 after induction of DNA breaks, a DNA damage-dependent association of BRCA1 with TOPBP1 has been revealed. These protein complexes of TOPBP1 and BRCA1 may be instrumental for driving productive HR, and will be evaluated in detail in this proposal. The third part of the proposal focuses on how post-translational modification by ubiquitination and SUMOylation of proteins at break sites affects BRCA1-mediated HR. In particular, RNF4, a ‘SUMO-targeted E3 ubiquitin ligase’, with a known role in DNA repair, will be the subject of further investigation. RNF4 mutations are found in breast cancer cells, often in association with mutations in the key cellular growth regulator, p53. Using a conditional knockout mouse model that was recently generated in this lab, in conjunction with mouse models of breast cancer and medulloblastoma, the importance of RNF4 for prevention of tumor formation will be measured. This research will deepen our understanding of the regulation of BRCA1 at DNA break sites, and identify potential targets and/or avenues to prevent mutations caused by defective DNA repair.

项目摘要：“招募 BRCA1 相关同源基因重组机械” 很大一部分肿瘤表现出由于 DNA 修复问题而产生的突变模式。关键 DNA 修复基因（例如 BRCA1、PALB2 和 BRCA2）的缺陷与效率低下有关通过同源重组 (HR) 进行 DNA 修复需要这些因素中的每一个来加载关键 HR。中间体，RAD51，位于 DNA 双链断裂位点 BRCA1 被募集到的确切机制。 DNA 断裂位点并被激活尚不完全清楚，目前尚未完全了解如何设计有效的疗法。由于缺乏对 BRCA1 功能的了解，抵消 DNA 修复缺陷就会受到损害。该提案的总体目标是建立一个集成模型来说明 BRCA1 如何本地化和通过研究控制 DNA 断裂位点的三种机制来实现这一目标。 BRCA1 相关修复机制的招募首先，BARD1 对 BRCA1 的贡献。 BARD1 是一种与 BRCA1 合作的 RING 结构域蛋白，并且似乎会评估招募情况。在 DNA 初步损伤后的早期时间点控制 BRCA1 向 DNA 断裂位点的募集。新型小鼠模型的结果表明，BARD1 与 BRCA1 的相互作用对于该提案中的工作将阐明 BARD1 控制 BRCA1 修复的机制。其次，将确定 TOPBP1 依赖的 BRCA1 募集机制。一种大型支架蛋白，可加载到 DNA 损伤位点的染色质上并招募多个 DNA 修复通过利用先进的蛋白质组学方法来研究BRCA1的蛋白质网络相互作用。诱导 DNA 断裂后，BRCA1 与 TOPBP1 的 DNA 损伤依赖性关联已被证实 TOPBP1 和 BRCA1 的这些蛋白质复合物可能有助于提高 HR 的生产力，并将在本提案的第三部分中详细评估如何翻译后。断裂位点蛋白质的泛素化和 SUMO 化修饰会影响 BRCA1 介导的 HR。特别是，RNF4，一种“SUMO 靶向 E3 泛素连接酶”，在 DNA 修复中具有已知的作用，将成为研究的主题进一步研究发现乳腺癌细胞中存在 RNF4 突变，通常与以下突变相关。关键的细胞生长调节剂 p53 使用最近生成的条件敲除小鼠模型。在本实验室中，结合乳腺癌和髓母细胞瘤小鼠模型，RNF4 的重要性这项研究将加深我们对预防肿瘤形成的认识。 DNA 断裂位点 BRCA1 的调控，并确定潜在的靶标和/或预防突变的途径由 DNA 修复缺陷引起。