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

描述（由申请人提供）：环境多环芳烃（PAHs），当被细胞代谢激活时，与DNA形成多种致突变和致癌的加合物。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加合物作出反应的关键细胞途径-足够全面，为识别多环芳烃暴露特别增加癌症风险的个体提供信息基础。我们假设，最终的净效率和保真度与这些相互关联的途径响应特定的DNA损伤决定其致突变性和致癌性。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，以及基因决定的个体对它们的反应效力。我们提出的探索性研究特别侧重于翻译DNA聚合酶（TLS pol）绕过多环芳烃加合物，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的协调。我们将研究经B[a] p -环氧二醇（B[a]PDE）处理的活体动物的mmr依赖性突变抑制和凋亡终点，以及导致这些终点的生化途径。特异性目的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诱导的突变频率和谱相关，并与B[a]PDE对转基因Msh2-/-和Msh2+/+结肠隐窝干细胞室细胞更新（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获得的人类核提取物来分析新型模型DNA底物的处理，这些底物含有与模板B[a]P-鸟嘌呤加合物密切相关的可引起切除的碱基错对（G/T）；因此，随后的DNA再合成必须绕过这些加合物。在核磁共振切除后的DNA再合成过程中，将开发加合物旁路效率的检测方法，并通过一种新的方法分析加合物下游DNA再合成的保真度。对tlspol Pol：和Pol7水平的调节或PCNA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在未来的综合研究中要解决的假设。

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.