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

• 批准号: 10599897
• 负责人: Samuel Bunting
• 金额: $ 38.31万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10599897
• 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; Methods; Microscopy; Modeling; Mus; Mutation; PALB2 gene; Pathologic; Pathway interactions; Pattern; Phenotype; Phosphorylation; Play; Poly(ADP-ribose) Polymerase Inhibitor; Post-Translational Protein Processing; Predisposition; Prevention; Productivity; 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; cancer type; cell growth; cohort; conditional knockout; design; effective therapy; homologous recombination; 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

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相关的同源物 重组机械” 大部分肿瘤显示出由于DNA修复问题而出现的突变模式。 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）修复DNA。每个因素都需要加载密钥HR DNA双链断裂位点中的中间rad51。招募BRCA1的确切机制 DNA断裂位点并被激活尚不完全了解。设计有效疗法的努力 缺乏对BRCA1功能的理解，抵消DNA修复缺陷受到了破坏。 该提案的总体目标是为BRCA1局部化和 在DNA断裂部位激活。通过研究控制的三种机制，将实现此目标 招募BRCA1相关的维修机械。首先，Bard1对BRCA1的贡献 将评估招聘。 Bard1是与BRCA1合作的环域蛋白质，似乎 在DNA损伤后的早期点，将BRCA1募集到DNA断裂位点。初步的 新型小鼠模型的结果表明，Bard1与BRCA1的相互作用对于 正常的DNA修复。该提案中的工作将阐明Bard1控制BRCA1维修的机制 活动。其次，将确定依赖TOPBP1的机理BRCA1的募集机理。 TopBp1是 大型脚手架蛋白，该蛋白质在DNA损伤位点上加载到染色质上，并报告多个DNA修复 因素。通过使用先进的蛋白质组学方法研究BRCA1的蛋白质相互作用网络 诱导DNA断裂后，BRCA1与TOPBP1的DNA损伤依赖性关联已是 揭示。这些TOPBP1和BRCA1的蛋白质复合物可能对驱动生产力的人力资源有助于 并将在本提案中详细评估。该提案的第三部分侧重于翻译后如何 通过泛素化和蛋白质在休息位点进行修饰会影响BRCA1介导的HR。在 特别是，RNF4是“靶向相关的E3泛素连接酶”，在DNA修复中具有已知作用，将是 进一步调查。 RNF4突变在乳腺癌细胞中发现，通常与突变有关 钥匙细胞生长调节剂p53。使用最近生成的条件敲除鼠标模型 在这个实验室中，与乳腺癌和髓母细胞瘤的小鼠模型一起，RNF4的重要性 为了预防肿瘤形成。这项研究将加深我们对 在DNA断裂位点调节BRCA1，并确定潜在的靶标和/或途径以防止突变 由DNA修复缺陷引起。