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.

项目总结：“BRCA 1相关同源基因的重新鉴定” 重组机” 相当大比例的肿瘤显示出由于DNA修复问题而出现的突变模式。 关键DNA修复基因如BRCA 1、PALB 2和BRCA 2的缺陷与效率低下有关。 同源重组（HR）DNA修复。这些因素中的每一个都需要加载关键的HR 中间体，RAD 51，在DNA双链断裂位点。BRCA 1被招募的确切机制 DNA断裂位点并被激活还没有完全理解。努力设计有效的治疗方法， 由于缺乏对BRCA 1功能的了解，抵消DNA修复缺陷的能力受到了破坏。 该提案的总体目标是为BRCA 1如何本地化提供一个综合模型， 在DNA断裂位点被激活。这一目标将通过研究控制细胞凋亡的三种机制来实现。 招募BRCA 1相关的修复机器。首先，BARD 1对BRCA 1的贡献 招聘将进行评估。BARD 1是一种与BRCA 1结合的RING结构域蛋白， 在DNA损伤后的早期时间点控制BRCA 1向DNA断裂位点的募集。初步 新的小鼠模型的结果表明，BARD 1与BRCA 1的相互作用是必不可少的， 正常的DNA修复这项提案中的工作将阐明BARD 1控制BRCA 1修复的机制 活动其次，将确定BRCA 1的TOPBP 1依赖性募集机制。TOPBP 1是 一种大的支架蛋白，在DNA损伤位点加载到染色质上并招募多个DNA修复 因素通过采用先进的蛋白质组学方法研究BRCA 1的蛋白质相互作用网络 在诱导DNA断裂后，BRCA 1与TOPBP 1的DNA损伤依赖性相关性已被证实。 揭示TOPBP 1和BRCA 1的这些蛋白质复合物可能有助于驱动生产性HR， 并将在本提案中详细评估。第三部分的建议主要集中在如何翻译后 蛋白质断裂位点的泛素化和SUMO化修饰影响BRCA 1介导的HR。 特别是，RNF 4，一种“SUMO靶向E3泛素连接酶”，在DNA修复中具有已知的作用，将成为本研究的主题。 进一步调查在乳腺癌细胞中发现了RNF 4突变，通常与以下突变相关： 关键的细胞生长调节因子p53使用一种条件性基因敲除小鼠模型， 在这个实验中，结合乳腺癌和髓母细胞瘤的小鼠模型， 用于预防肿瘤形成。这一研究将加深我们对 BRCA 1在DNA断裂位点的调控，并确定潜在的靶点和/或预防突变的途径 是由DNA修复缺陷引起的