HOMOLOGY-DIRECTED DNA REPAIR PROTEINS BRCA2 AND RAD51 IN TUMOR RELEVANT TISSUES

肿瘤相关组织中同源定向 DNA 修复蛋白 BRCA2 和 RAD51

• 批准号: 8686532
• 负责人: Maria Jasin
• 金额: $ 36.24万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8686532
• 关键词: Address; Animals; BRCA2 Mutation; BRCA2 Protein; BRCA2 gene; Breast Epithelial Cells; C-terminal; Cell Culture Techniques; Cell Line; Cell Maintenance; Cell Survival; Cells; Chromosomal Instability; Complement; DNA Damage; DNA Repair Pathway; DNA Sequence Rearrangement; DNA repair protein; Development; Disease; Embryo; Fanconi' s Anemia; Filament; Funding; Genetic; Genetic Recombination; Genome; Genomic Instability; Genomics; Germ Cells; Germ-Line Mutation; Histocompatibility Testing; Human; Lesion; Location; Loss of Heterozygosity; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Modeling; Mus; Mutagens; Mutation; Pathway interactions; Patients; Phenotype; Physiological; Property; Proteins; Reporter; Role; Sequence Homologs; Site; Somatic Cell; Stem cells; Structure; Tissues; Transgenic Mice; Tumor Suppression; Tumor Suppressor Proteins; cell type; chromosome loss; developmental disease; homologous recombination; malignant breast neoplasm; mammary epithelium; mouse genome; mouse model; mutant; novel; paralogous gene; parent grant; public health relevance; recombinase; repaired; research study; tumor; tumorigenesis

PROJECT SUMMARY/ABSTRACT Homologous recombination, also called homology-directed repair (HDR), is a major DNA repair pathway for lesions such as double-strand breaks (DSBs). Several proteins critical to the HDR pathway have been identified as tumor suppressors. Notable among these is the breast cancer suppressor BRCA2 which interacts with and promotes the function of the RAD51 recombinase, the critical protein for strand exchange between homologous sequence. BRCA2 mutations are also associated with the developmental disorder Fanconi anemia. The long-term objective is to understand the role of HDR proteins in tumor-relevant tissue types, as HDR deficiency is a major target for tumor therapies under development. The specific aims are: 1. To investigate roles of HDR proteins in human mammary epithelial cells. Genetic loss of HDR proteins results in embryonic lethality in the mouse, but mammary tumors form in conditional mouse models as in patients with germline mutations. To understand the cellular roles of HDR proteins in the tumor-relevant cell type, we plan to construct isogenic human mammary epithelial cell lines with mutations in the HDR proteins BRCA2, RAD51, and select RAD51 paralogs. A major question to be addressed is whether loss of any one of the HDR proteins results in a cell lethal phenotype. For mutants that are inviable, rescue experiments will be attempted. Mutants that are viable will be interrogated for a number of properties, including HDR deficiency, chromosome instability, and sensitivity to DNA damaging agents. In limited cases, epistatic relationships between HDR proteins will be determined. 2. To determine the requirement for RAD51 and BRCA2 in mouse mammary epithelial cells. As a complement to Aim1 plan to delete BRCA2 from primary mouse mammary epithelial cell cultures in the presence or absence of p53 to determine whether BRCA2 is required for cellular viability. We will also develop a conditional RAD51 deletion model to address its requirement for somatic cell viability and tumor suppression. 3. To determine the role of the BRCA2 C terminus in HDR, replication fork protection, and genome integrity. BRCA2 interaction with RAD51 at a C terminal site has been implicated in stabilizing RAD51 filaments, although the BRCA2 C terminus may have additional functions which promote HDR. We plan to investigate HDR in cells and tissues from mice deleted for the BRCA2 C terminus using a novel transgenic mouse model. We will also address whether loss of heterozygosity is increased in these mice, and whether they are susceptible to endogenous genotoxins similar to Fanconi anemia mice. Germ cell development will also be examined as a model for stem cell maintenance and recombination. RAD51 filament stabilization by BRCA2 was recently found by our lab to be of critical importance for the protection of stalled replication forks from being degraded. A separation of function mutation in BRCA2 will be developed to determine the physiological effects of loss of replication fork protection.

项目摘要/摘要 同源重组，也称为同源指导修复（HDR），是主要的DNA修复 诸如双链断裂（DSB）等病变的途径。对HDR途径至关重要的几种蛋白质具有 被确定为肿瘤抑制剂。其中值得注意的是抑制乳腺癌的BRCA2 与Rad51重组酶（用于链交换的关键蛋白质）的功能相互作用并促进 在同源序列之间。 BRCA2突变也与发育障碍有关 Fanconi贫血。长期目标是了解HDR蛋白在肿瘤与肿瘤相关组织中的作用 类型，由于HDR缺乏是开发肿瘤疗法的主要靶点。具体目的是： 1。研究HDR蛋白在人类乳腺上皮细胞中的作用。 HDR的遗传丧失 蛋白质会导致小鼠的胚胎致死性，但有条件小鼠模型中形成乳腺肿瘤作为 在种系突变的患者中。了解HDR蛋白在肿瘤相关细胞中的细胞作用 类型，我们计划构建具有HDR蛋白突变的同源性人类乳腺上皮细胞系 BRCA2，RAD51和选择RAD51旁系同源物。要解决的一个主要问题是 HDR蛋白会导致细胞致死表型。对于不可或缺的突变体，救援实验将是 尝试。可行的突变体将对许多属性（包括HDR缺乏症）审问， 染色体不稳定性和对DNA破坏剂的敏感性。在有限的情况下，上皮关系 将确定HDR蛋白之间。 2。确定小鼠乳腺上皮细胞中RAD51和BRCA2的需求。作为 补充AIM1计划从原发性小鼠乳腺上皮细胞培养物中删除BRCA2 p53的存在或不存在，以确定是否需要BRCA2才能使细胞生存能力。我们还将发展 有条件的RAD51缺失模型，以解决其对体细胞活力和抑制肿瘤的需求。 3。确定BRCA2 C末端在HDR中的作用，复制叉保护和基因组 正直。 BRCA2与C端位点处的RAD51相互作用与稳定RAD51有关 细丝，尽管BRCA2 C末端可能具有促进HDR的其他功能。我们计划 使用新型转基因研究细胞和组织中的细胞和组织中的HDR 鼠标模型。我们还将解决这些小鼠的杂合性丧失是否增加，以及是否是否 它们容易受到类似于范科尼贫血小鼠的内源性基因毒素的影响。生殖细胞发育将 也可以作为干细胞维持和重组的模型进行检查。 RAD51细丝稳定 最近，我们的实验室发现BRCA2对于保护停滞的复制叉非常重要 从退化。将开发BRCA2中功能突变的分离，以确定 损失复制叉保护的生理影响。