Mechanism of RAD51C fork protection and environmental carcinogenesis

摘要 环境致癌物质会造成DNA损伤，从而阻碍DNA复制。这会危及基因组 诚信对于多样化的疾病抑制至关重要。越来越多的报告表明，携带基因突变的个体 乳腺癌易感基因(BRCA)(在150多人中发现1例)增加了癌症发病率 暴露在环境致癌物质中。造成这种情况的根本原因最近引起了人们的争论。 在这个应用中，我们将确定dna复制叉保护(Fp)的分子机制。 由BRCA3/RAD51C介导以及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也是最不为人所知的成员。 由于与RAD51的序列同源性，RAD51在同源定向的双基因表达过程中受BRCA1/2调控。 链(DSB)断裂修复，RAD51C的研究主要集中在其修复功能上。然而，患者数据 提示具有额外的肿瘤抑制功能；10例与疾病相关的RAD51C患者中有8例突变 最初确定的，不会造成DSB-修复缺陷，并在此基础上被指定为未分类。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 疾病外显的意义。BRCA基因除了具有DNA修复功能外，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Mre11核酸酶的降解，一种新的功能 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可防止基因组不稳定 在几乎所有环境致癌物引发的停滞不前的DNA复制分叉上无处不在。因此，我们 假设BRCA3/RAD51C通过保护失速的 DNA复制分叉。在目标1)中，我们将定义RAD51C介导的叉子稳定性的机制，启用 通过我们发现和了解新的BRCA基因在FP中的功能，通过我们开发的单细胞 通过对RAD51C DNA结构的理解，检测蛋白质-DNA复制叉相互作用(SIRF) 结合和ATPase结构域以及建立CRISPR/Cas9敲入突变体RAD51C人细胞 台词。在目标2)中，我们将确定FP缺陷是否会促进环境致癌物诱发的乳房 体内肿瘤的发生，这是因为我们已经建立了一个具有FP基因的活体突变RAD51C小鼠模型 缺陷，但没有明显的DSB修复缺陷，根据我们对FP和DSB的遗传控制的理解 PTIP和53BP1，这使我们能够从基因上检测和区分FP和DSB修复贡献 致癌物诱导的乳腺癌发生。总的来说，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和肿瘤。 除了BRCA基因外，现在已知还有许多基因控制FP，我们的研究结果可能有 对一大群人进行准确和有效的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.