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

描述（由申请人提供）：环境中的多环芳烃（PAH）在被细胞代谢激活时，与DNA形成各种致突变和致癌加合物。细胞反应的特征仍然很差。该双PI项目的长期目标是了解对PAH衍生的DNA加合物做出反应的关键细胞途径-足够全面，以提供用于识别PAH暴露特别增加癌症风险的个体的信息基础。我们推测，这些相互关联的途径对特定DNA损伤的最终净效率和特异性决定了它们的致突变性和致癌性。因此，人类暴露于多环芳烃的风险取决于DNA中诱导的特定加合物，以及个体对它们的遗传反应。我们提出的探索性研究特别关注跨病变DNA聚合酶（TLS pols）绕过PAH加合物的协调，并通过错配修复（MMR）纠正这种绕过产生的靶向和非靶向复制错误。我们将研究用B[a] β-二醇环氧化物（B[a]PDE）处理的活动物中的MMR依赖性突变抑制和细胞凋亡终点，以及导致这些终点的生化途径。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诱导的突变的频率和谱以及B[a]PDE对转基因Msh 2-/-与Msh 2 +/+的结肠隐窝的干细胞隔室中的细胞更新（细胞复制和凋亡）的影响相关。具体目标2是使用复杂但生物化学上可接近的人核提取物来分析新模型DNA底物的加工，所述新模型DNA底物含有与模板B[a]P-鸟嘌呤加合物紧密相邻的引起切除的碱基错对（G/T）;因此，这样的加合物必须被随后的DNA再合成绕过。将开发用于在MMR切除后DNA再合成期间旁路加合物的效率的测定，并且将通过一种新方法分析加合物下游DNA再合成的保真度。将确定TLS pols Pol：和Pol 7的水平或PCNA泛素化的调节的效果。这两个相互关联的目标的结果将指导在未来的综合研究中要解决的假设。 公共卫生相关性：这项探索性工作将为今后更广泛的研究提供基础。这些将有助于识别因环境因素诱发的癌症风险增加的个体。

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.