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.

项目总结/摘要 同源重组，也称为同源定向修复（HDR），是一种主要的DNA修复 损伤途径，如双链断裂（DSB）。HDR途径的几种关键蛋白质 被鉴定为肿瘤抑制剂。其中值得注意的是乳腺癌抑制因子BRCA 2， 与RAD 51重组酶相互作用并促进其功能，RAD 51重组酶是链交换的关键蛋白 同源序列之间。BRCA 2突变也与发育障碍有关 范可尼贫血。长期目标是了解HDR蛋白在肿瘤相关组织中的作用， 类型，因为HDR缺陷是正在开发的肿瘤治疗的主要目标。具体目标是： 1.探讨HDR蛋白在人乳腺上皮细胞中的作用。HDR的基因丢失 蛋白质导致小鼠胚胎死亡，但乳腺肿瘤在条件小鼠模型中形成， 生殖细胞突变的患者。了解HDR蛋白在肿瘤相关细胞中的细胞作用 型，我们计划构建HDR蛋白突变的同基因人乳腺上皮细胞系 BRCA 2、RAD 51和选择RAD 51旁系同源。需要解决的一个主要问题是， HDR蛋白导致细胞致死表型。对于无法存活的变种人， 企图。将询问有活力的突变体的许多特性，包括HDR缺陷， 染色体不稳定性和对DNA损伤剂的敏感性。在有限的情况下，上位关系 将确定HDR蛋白之间的关系。 2.确定小鼠乳腺上皮细胞对RAD 51和BRCA 2的需求。作为 补充Aim 1计划从原代小鼠乳腺上皮细胞培养物中删除BRCA 2， p53的存在或不存在，以确定BRCA 2是否是细胞活力所必需的。我们还将开发 条件性RAD 51缺失模型，以解决其对体细胞活力和肿瘤抑制的需求。 3.确定BRCA 2 C末端在HDR、复制叉保护和基因组中的作用 完整BRCA 2与RAD 51在C末端位点的相互作用涉及稳定RAD 51 丝，虽然BRCA 2 C末端可能有额外的功能，促进HDR。我们计划 使用新型转基因研究BRCA 2 C末端缺失小鼠细胞和组织中的HDR 小鼠模型我们还将讨论这些小鼠中杂合性丢失是否增加，以及是否 它们对类似于范可尼贫血小鼠的内源性基因毒素敏感。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。RAD 51灯丝稳定， 我们的实验室最近发现BRCA 2对于保护停滞的复制分叉至关重要 被降级。将开发BRCA 2中功能突变的分离，以确定 复制叉保护丧失的生理影响。