A Novel Role for the Fanconi Anemia Pathway in Replication of B[a]P-Adducted DNA

描述（由申请方提供）：本提案的广泛长期目标是阐明范可尼贫血（FA）通路在保护细胞免受苯并[a]芘（B[a]P）诱导的DNA损伤的致癌作用中的作用。B[a]P是一种丰富且普遍存在的环境致癌物，其在细胞内代谢产生苯并[a]芘二氢二醇环氧化物（BPDE）。BPDE诱导的DNA损伤触发Rad 18（一种E3连接酶）依赖的PCNA单泛素化和特异性DNA聚合酶Pol kappa向复制叉的募集。与在DNA损伤位点停滞的复制型DNA聚合酶相比，Pol kappa可以使用BPDE加合的DNA作为模板进行DNA合成。Pol κ介导的TLS使细胞能够耐受BPDE加合物的致突变和致死作用，从而有助于基因组稳定性和肿瘤抑制。然而，Pol κ调节的分子机制尚未完全了解。我们的初步研究表明，范可尼贫血（FA）蛋白密切参与Rad 18和Pol κ介导的TLS的B[a]P病变。本研究的具体目的是：（1）确定BPDE诱导的FANCD 2（FA途径的假定效应子）与Pol kappa之间相互作用的分子基础。(2)确定Rad 18激活FA通路的机制。(3)测试FA和TLS途径的相互依赖性。目的1将突变Pol κ中的泛素结合基序，并确定这些突变对FANCD 2缔合的影响。此外，我们将测试Pol kappa和FA通路相互作用蛋白REV 1在介导Pol kappa-FANCD 2相互作用中的作用。目标2将测试Rad 18是否直接或通过调节上游FA核心复合物组分来修饰FANCD 2。目的3将确定FANC缺陷是否会损害BPDE病变的TLS，相反，缺陷的TLS是否会影响BPDE对FA通路的激活。我们的研究结果将提供一个新的模式的机制，FA途径是协调与TLS酶在响应环境B[a]P（和可能的其他遗传毒素），以保持基因组的稳定性。我们的研究可能有助于识别环境B[a] P诱导疾病的高风险个体。此外，我们的工作可能会导致癌症治疗的新药物靶点：与B[a]P类似，许多化疗药物具有遗传毒性并激活检查点途径。我们已经表明，Pol κ-或Rad 18-缺陷使细胞对B[a] P诱导的死亡敏感。潜在地，靶向FA组分、Rad 18、Pol κ或其他TLS酶的小分子可以使癌细胞对化疗剂的杀伤敏感。公共卫生相关性： 苯并[a]芘（B[a]P）是一种丰富的环境污染物，它会破坏DNA，从而导致遗传密码的永久性改变（称为“突变”），从而导致癌症。我们的研究结果表明，一组细胞蛋白质称为“范可尼贫血”蛋白（FANCs）发挥作用，防止环境B[a]P-诱导的突变。拟议的实验旨在了解FANCs帮助细胞耐受环境诱导的DNA损伤并预防癌症的机制。

DESCRIPTION (provided by applicant): The broad long-term objective of this proposal is to elucidate the role of the Fanconi Anemia (FA) pathway in protecting cells from the carcinogenic effects of Benzo[a]pyrene (B[a]P)-induced DNA damage. B[a]P is an abundant and ubiquitous environmental carcinogen that is metabolized intracellularly to generate Benzo[a]pyrene Di-hydrodiol-Epoxide (BPDE). BPDE-induced DNA damage triggers Rad18 (an E3 ligase)- dependent mono-ubiquitination of PCNA and recruitment of the specialized DNA polymerase Pol kappa to replication forks. In contrast with replicative DNA polymerases which stall at sites of DNA damage, Pol kappa can perform DNA synthesis using BPDE-adducted DNA as a template. Pol kappa-mediated TLS enables cells to tolerate the mutagenic and lethal effects of BPDE adducts, thereby contributing to genomic stability and tumor suppression. However, the molecular mechanism(s) of Pol kappa regulation are incompletely understood. Our preliminary studies suggest that the Fanconi Anemia (FA) proteins are intimately involved in Rad18 and Pol kappa- mediated TLS of B[a]P lesions. The Specific Aims of this proposal are: (1) To determine the molecular basis for BPDE-induced interaction between FANCD2 (the putative effector of the FA pathway) and Pol kappa. (2) To determine the mechanism by which Rad18 activates the FA pathway. (3) To test the interdependence of the FA and TLS pathways. Aim 1 will mutate ubiquitin binding motifs in Pol kappa and determine the effect of these mutations on FANCD2 association. Additionally, we will test the role of the Pol kappa- and FA pathway-interacting protein REV1 in mediating Pol kappa-FANCD2 interactions. Aim 2 will test whether Rad18 modifies FANCD2 directly or via regulation of upstream FA core complex components. Aim 3 will determine whether FANC- deficiency compromises TLS of BPDE lesions and conversely, whether defective TLS affects activation of the FA pathway by BPDE. Results of our studies will provide a novel paradigm for mechanisms by which the FA pathway is coordinated with TLS enzymes in response to environmental B[a]P (and possibly other genotoxins) to maintain genomic stability. Our studies might help identify individuals that are at high-risk for environmental B[a]P-induced disease. Moreover, our work could lead to novel drug targets for cancer therapy: Similar to B[a]P, many chemotherapies are genotoxic and activate checkpoint pathways. We have shown that Pol kappa- or Rad18-deficiency sensitizes cells to B[a]P-induced death. Potentially, small molecules that target FA components, Rad18, Pol kappa, or other TLS enzymes could sensitize cancer cells to killing by chemotherapeutic agents. PUBLIC HEALTH RELEVANCE: Benzo[a]pyrene (B[a]P) is an abundant environmental pollutant that damages DNA thereby causing permanent changes to the genetic code (termed 'mutations') that can result in cancer. Our results suggest that a group of cellular proteins termed the 'Fanconi Anemia' proteins (FANCs) play a role in preventing environmental B[a]P- induced mutations. The proposed experiments aim to understand the mechanisms by which FANCs help cells tolerate the effects of environmentally-induced DNA damage and protect against cancer.