Mechanism of RAD51C fork protection and environmental carcinogenesis

总结 环境致癌物会造成DNA损伤，阻碍DNA复制。这危害了基因组 完整性对多种疾病的抑制至关重要。越来越多的报告显示， 乳腺癌易感基因（BRCA）（在150人中发现超过1人）增加了癌症发病率 暴露在环境致癌物中。这一现象的根本原因最近引起了争论。 在这个应用中，我们将确定DNA复制叉保护（FP）的分子机制 BRCA3/RAD51C介导的肿瘤发生，以及RAD51C和FP如何抑制致癌物诱导的肿瘤发生。 RAD51C是BRCA疾病抑制基因家族中最新的成员，也是人们了解最少的成员。 由于与RAD51的序列同源性，RAD51在同源性定向的双- 由于RAD51C是双链断裂（DSB）修复的重要载体，因此RAD51C的研究主要集中在其修复功能上。然而，患者数据 提示额外的肿瘤抑制功能; 10例与疾病相关的RAD51C患者突变中有8例 最初确定，不会导致DSB维修缺陷，并基于此被指定为未分类。然而， 随后的多项独立乳腺癌人群研究发现了相同的等位基因，这表明 对疾病转归的意义。BRCA基因除了DNA修复外还具有细胞功能。重要的是这 包括保护停滞的DNA复制叉免受MRE 11核酸酶的降解，MRE 11核酸酶是一种新的功能性 我们最近定义的路径。令人兴奋的是，我们的初步数据显示许多未分类的癌症- 相关的RAD51C突变损害FP，与DSB修复无关。FP防止基因组不稳定性 几乎所有的环境致癌物都能引起DNA复制叉的停滞。我们因此 假设BRCA3/RAD51C通过保护停滞细胞来保护环境致癌物 DNA复制叉。在目标1）中，我们将定义RAD51C介导的分叉稳定性的机制， 通过我们对FP中新的BRCA基因功能的发现和理解，通过我们对单细胞 蛋白质-DNA复制叉相互作用（SIRF）的测定，通过我们对RAD51C DNA结构的理解 结合结构域和ATP酶结构域，并通过建立CRISPR/CAS9敲入突变体RAD51C人细胞 线在目标2）中，我们将确定FP缺陷是否促进环境致癌物诱导的乳腺癌。 体内肿瘤发生，我们已经建立了一个可行的突变体RAD 51 C小鼠模型与FP 缺陷，但没有明显的DSB修复缺陷，并通过我们对FP和DSB的遗传控制的理解， PTIP和53BP1，这使我们能够从基因上测试和区分FP和DSB修复的贡献， 致癌物诱发的乳腺癌。总体而言，拟议的研究将提供基本的 了解RAD51C介导的FP如何抑制环境诱导的基因组不稳定性和肿瘤。 由于目前已知除了BRCA基因之外还有许多基因控制FP，因此我们的研究结果可能具有以下意义： 这对准确和有效地对一大群人进行疾病风险评估具有重要的广泛意义。

SUMMARY Environmental carcinogens generate DNA damage that stalls DNA replication. This jeopardizes genomic integrity critical to diverse disease-suppression. Accumulating reports show that individuals with mutations in breast cancer predisposition genes (BRCA) (found in more than 1 in 150 people) have increased cancer rates upon exposure to environmental carcinogens. The underlying cause for this has recently come under debate. In this application, we will determine the molecular mechanism of DNA replication fork protection (FP) mediated by BRCA3/RAD51C and how RAD51C and FP suppress carcinogen-induced tumorigenesis. RAD51C is the newest and perhaps least understood member of the BRCA disease suppressor family. Because of sequence homology to RAD51, which is regulated by BRCA1/2 during homology-directed double- strand (DSB) break repair, RAD51C studies have focused on its repair function. However, patient data suggests an additional tumor suppression function; 8 out of 10 disease-linked RAD51C patient mutations initially identified, do not cause DSB-repair deficiencies and based on this were designated unclassified. Yet, multiple subsequent independent breast cancer population studies identified the same alleles, suggesting significance for disease penetrance. BRCA genes have cellular functions besides DNA repair. Importantly, this includes the protection of stalled DNA replication forks from degradation by MRE11 nuclease, a new functional pathway that we have recently defined. Excitingly, our preliminary data shows many of the unclassified cancer- associated RAD51C mutations compromise FP, irrespective of DSB-repair. FP prevents genome instability ubiquitously at stalled DNA replication forks as induced by virtually all environmental carcinogens. We thus hypothesize that BRCA3/RAD51C safeguards against environmental carcinogens through protection of stalled DNA replication forks. In Aim 1) we will define the mechanism of RAD51C mediated fork stability, enabled by our discovery and understanding of new BRCA gene functions in FP, by our development of a single-cell assay for protein-DNA replication fork interactions (SIRF), by our structural understanding of the RAD51C DNA binding and ATPase domains and by having established CRISPR/CAS9 knock-in mutant RAD51C human cell lines. In Aim 2) we will determine if FP defects promote environmental carcinogen-induced mammary tumorigenesis in vivo, enabled by our having established a viable mutant RAD51C mouse model with FP defects, but no apparent DSB-repair defects, and by our understanding of genetic control of FP and DSBs by PTIP and 53BP1, that allows us to genetically test and distinguish FP from DSB repair contributions to carcinogen-induced mammary carcinogenesis. Collectively the proposed research will provide fundamental knowledge of how RAD51C-mediated FP suppresses environmentally induced genome instability and tumors. As many genes besides BRCA genes are now known to control FP, the outcome of our studies can have important broad implications for accurate and efficient disease-risk assessment for a large group of people.