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

项目摘要/摘要 同源重组，也被称为同源定向修复(Hdr)，是一种主要的DNA修复 损伤的途径，如双链断裂(DSB)。几种对HDR途径至关重要的蛋白质 被确认为肿瘤抑制因子。其中值得注意的是乳腺癌抑制基因BRCA2 与RAD51重组酶相互作用并促进其功能，RAD51重组酶是链交换的关键蛋白质 在同源序列之间。BRCA2突变也与发育障碍有关 范科尼贫血。长期目标是了解HDR蛋白在肿瘤相关组织中的作用 由于HDR缺乏症是正在开发的肿瘤治疗的主要靶点。具体目标是： 1.探讨HDR蛋白在人乳腺上皮细胞中的作用。人类发育迟缓的遗传缺失 蛋白质导致小鼠胚胎死亡，但在有条件的小鼠模型中，乳腺肿瘤形成为 在有生殖系突变的患者中。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蛋白突变的等基因人乳腺上皮细胞系 BRCA2、RAD51和SELECT RAD51 Paralog。一个需要解决的主要问题是，如果失去任何一个 HDR蛋白导致细胞致死表型。对于无法存活的变种人，救援实验将是 已尝试。可以存活的突变体将被询问一系列特性，包括HDR缺陷， 染色体不稳定，以及对DNA损伤剂的敏感性。在有限的情况下，上位关系 HDR蛋白之间的相互作用将被确定。 2.确定小鼠乳腺上皮细胞对RAD51和BRCA2的需求。作为一名 对Aim1计划的补充：从原代培养的小鼠乳腺上皮细胞中删除BRCA2 P53的存在或不存在来确定BRCA2是否是细胞存活所必需的。我们还将发展 一个有条件的RAD51缺失模型，以满足其对体细胞活性和肿瘤抑制的要求。 3.确定BRCA2C末端在HDR、复制分叉保护和基因组中的作用 正直。BRCA2与RAD51在C末端的相互作用与稳定RAD51有关 细丝，尽管BRCA2C末端可能具有促进HDR的额外功能。我们计划 利用一种新的转基因技术研究BRCA2C末端缺失的小鼠细胞和组织中的HDR 老鼠模型。我们还将讨论这些小鼠的杂合性丢失是否增加，以及 它们对内源性基因毒素的敏感性类似于范科尼贫血小鼠。生殖细胞发育将 也可以被视为干细胞维持和重组的模型。RAD51长丝稳定化 我们的实验室最近发现BRCA2对于保护停滞的复制分叉至关重要 不会被降级。BRCA2中功能突变的分离将被开发来确定 失去复制分叉保护的生理效应。

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.