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

描述（由申请人提供）：同源重组，也称为同源定向修复（HDR），是损伤（如双链断裂（DSB））的主要DNA修复途径。几种对HDR途径至关重要的蛋白质已被鉴定为肿瘤抑制因子。其中值得注意的是乳腺癌抑制因子BRCA2，它与RAD 51重组酶相互作用并促进其功能，RAD 51重组酶是同源序列之间链交换的关键蛋白质。BRCA2突变也与发育障碍范可尼贫血有关。长期目标是了解HDR蛋白在肿瘤相关组织类型中的作用，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.目的探讨人类乳腺上皮细胞HDR蛋白的功能。HDR蛋白的遗传缺失导致小鼠胚胎死亡，但乳腺肿瘤在条件小鼠模型中形成，如在具有生殖系突变的患者中。为了了解HDR蛋白在肿瘤相关细胞类型中的细胞作用，我们计划构建HDR蛋白BRCA2、RAD51突变的同基因人乳腺上皮细胞系，并选择RAD51旁系同源物。要解决的一个主要问题是，任何一种HDR蛋白的丢失是否会导致细胞致死表型。对于无法存活的突变体，将尝试进行拯救实验。将询问存活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，将确定HDR蛋白之间的上位关系。 2.确定小鼠乳腺上皮细胞对RAD51和BRCA2的需求。作为对Aim1的补充，计划在存在或不存在p53的情况下从原代小鼠乳腺上皮细胞培养物中删除BRCA 2，以确定BRCA 2是否是细胞活力所必需的。我们还将开发一种条件性RAD51缺失模型，以解决其对体细胞活力和肿瘤抑制的要求。 3.确定BRCA2 C末端在HDR、复制叉保护和基因组完整性中的作用。BRCA2与RAD 51在C末端位点的相互作用已经涉及稳定RAD 51丝，尽管BRCA2 C末端可能具有促进HDR的额外功能。我们计划使用一种新的转基因小鼠模型研究BRCA2 C末端缺失小鼠的细胞和组织中的HDR。我们还将讨论这些小鼠的杂合性丢失是否增加，以及它们是否对类似于范可尼贫血小鼠的内源性基因毒素敏感。生殖细胞发育也将作为干细胞维持和重组的模型进行研究。我们的实验室最近发现，BRCA2对RAD51细丝的稳定性对于保护停滞的复制叉不被降解至关重要。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效应。

DESCRIPTION (provided by applicant): 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 germ line 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.