MMR-Coupled Translesion DNA Synthesis During Suppression of PAH-Induced Mutation

描述(申请人提供)：环境多环芳烃(PAHs)，当被细胞新陈代谢激活时，与DNA形成各种诱变和致癌加合物。细胞反应的特征仍然很差。这个双PI项目的长期目标是了解对多环芳烃衍生的DNA加合物做出反应的关键细胞途径--足够全面，足以为识别暴露于多环芳烃尤其会增加癌症风险的个人提供信息基础。我们假设这些相互连接的通路对特定DNA损伤的最终净效率和保真度决定了它们的致突变性和致癌性。因此，人类接触多环芳烃的风险取决于DNA中诱导的特定加合物，以及个体对这些加合物的遗传决定的反应能力。我们拟议的探索性研究具体集中在跨病变DNA聚合酶(TLS Pols)对PAH加合物的旁路与错配修复(MMR)对此类旁路产生的靶向和非靶向复制错误的纠正的协调。我们将研究B[a]-二醇环氧化物(B[a]PDE)处理的活体动物中依赖MMR的突变抑制和凋亡终点，以及通向这些终点的生化途径(S)。具体目的1是确定MMR缺乏对转基因小鼠完整组织中B[a]PDE诱导的遗传毒性的影响。立体特异性DNA加合物的水平将与B[a]PDE诱发突变的频率和光谱以及B[a]PDE对转基因Msh2-/-对Msh2/的结肠隐窝干细胞间细胞周转(细胞复制和凋亡)的影响相关。具体目标2是使用复杂但可生化访问的人核提取液来分析新型DNA模型底物的加工过程，该底物包含与模板B[a]P-鸟嘌呤加合物紧密相邻的引发切除的碱基错对(G/T)；因此，这些加合物必须被后续的DNA重新合成所绕过。将开发在MMR切除后DNA再合成过程中绕过加合物的效率的测试，并将用一种新的方法分析紧接在加合物下游的DNA再合成的保真度。TLS Pols Pol：和Pol7水平的调节或增殖细胞核抗原泛素化的影响将被确定。这两个相互关联的目标的结果将指导假说在未来的全面研究中得到解决。 公共卫生相关性：这项探索性工作将为未来更多、更广泛的研究提供基础。这些将有助于识别由环境因素引起的癌症风险增加的个人。

DESCRIPTION (provided by applicant): Environmental polycyclic aromatic hydrocarbons (PAHs), when activated by cellular metabolism, form a variety of mutagenic and carcinogenic adducts with DNA. Cellular responses remain poorly characterized. The long- term goal of this dual-PI project is an understanding of the key cellular pathways that respond to PAH-derived DNA adducts - comprehensive enough to provide an information base for identification of individuals for whom PAH exposures particularly increase cancer risk. We hypothesize that the final net efficiencies and fidelities with which these interconnected pathways respond to specific DNA lesions determine their mutagenicity and carcinogenicity. Thus, risks of human exposure to PAHs depend on the particular adducts induced in DNA, and the genetically-determined potencies of responses to them in individuals. Our proposed exploratory research focuses specifically on coordination of bypass of PAH adducts by translesion DNA polymerases (TLS pols) with correction by mismatch-repair (MMR) of targeted and untargeted replication errors generated by such bypass. We will investigate MMR-dependent mutation-suppression and apoptosis endpoints in live animals treated with B[a]P-diol epoxide (B[a]PDE), and the biochemical pathway(s) leading to these endpoints. Specific Aim 1 is to determine the effects of MMR deficiency on B[a]PDE-induced genotoxicity in intact tissues in transgenic mice. Levels of stereospecific DNA adducts will be correlated to frequencies and spectra of mutations induced by B[a]PDE and to B[a]PDE effects on cellular turnover (cell replication and apoptosis) in the stem cell compartment of colonic crypts of transgenic Msh2-/- versus Msh2+/+. Specific Aim 2 is to use complex but biochemically accessible human nuclear extracts to analyze processing of novel model DNA substrates that contain excision-provoking base-mispairs (G/T) that are closely adjacent to template B[a]P- guanine adducts; such adducts must thus be bypassed by subsequent DNA resynthesis. Assays for the efficiency of bypass of adducts during post-MMR-excision DNA resynthesis will be developed, and fidelity of DNA resynthesis immediately downstream of the adducts will be analyzed by a novel method. The effects of modulation of the levels of TLS pols Pol: and Pol7 or of PCNA ubiquitination will be determined. The outcomes of the two interconnected aims will direct the hypotheses to be addressed in future comprehensive studies. PUBLIC HEALTH RELEVANCE: This exploratory work will provide the basis for more future more extensive studies. These will help identify individuals at increased risk for cancer induced by environmental agents.