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

项目摘要：“招聘与BRCA1有关的同系人 重组机械“ 相当大比例的肿瘤表现出突变模式，这些模式是由于DNA修复问题而产生的。 关键DNA修复基因的缺失，如BRCA1、PALB2和BRCA2，与低效相关 DNA修复的同源重组(HR)。这些因素中的每一个都需要加载关键的HR 中间体，RAD51，位于DNA双链断裂位点。BRCA1被招募到的确切机制 DNA断裂部位并被激活，目前还不完全清楚。努力设计有效的治疗方法 由于缺乏对BRCA1功能的了解，中和DNA修复缺陷被破坏。 这项提议的总体目标是为BRCA1如何本地化和 在DNA断裂部位被激活。这一目标将通过调查三种控制 招募与BRCA1相关的维修机械。首先，BARD1对BRCA1的贡献 将对招聘进行评估。BARD1是一种环域蛋白，与BRCA1配对，似乎 在DNA损伤后的早期时间点控制BRCA1向DNA断裂部位的募集。初步 新的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。本提案中的工作将阐明BARD1控制BRCA1修复的机制 活动。其次，将确定依赖TOPBP1的BRCA1招募机制。TOPBP1为 一种大的支架蛋白，在DNA损伤部位加载到染色质上，并招募多个DNA修复 各种因素。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 在诱导DNA断裂后，BRCA1与TOPBP1之间存在DNA损伤依赖的关联 被揭露了。TOPBP1和BRCA1的这些蛋白质复合体可能有助于推动生产力HR， 并将在这份提案中进行详细评估。提案的第三部分侧重于后翻译 断裂位点蛋白质的泛素化和SUMO化修饰会影响BRCA1介导的HR。在……里面 特别是，RNF4，一种以相扑为靶点的E3泛素连接酶，在DNA修复中具有已知的作用，将成为 进一步调查。在乳腺癌细胞中发现RNF4突变，通常与 关键的细胞生长调节因子，P53。使用最近生成的条件性基因敲除小鼠模型 在这个实验室中，结合乳腺癌和髓母细胞瘤的小鼠模型，RNF4的重要性 对于预防肿瘤的形成将进行测量。这项研究将加深我们对 调节DNA断裂位点的BRCA1，并识别潜在的靶点和/或防止突变的途径 是由DNA修复缺陷引起的。

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.