Impact of PAH-DNA Lesions on DNA Repair and Replication

描述（由申请人提供）：人群暴露于各种环境致癌污染物，包括化石燃料燃烧产物和香烟烟雾中的物质。海湾地区的苯并[a]芘（B[a]P）是一类潜在致癌化合物多环芳烃（PAH）的最著名代表。与其他PAH物质一样，B[a]P被代谢活化为高反应性和致突变性B[a]P二环氧化物，与DNA反应形成加合物。低效的DNA修复和由聚合酶催化的DNA加合物的translesion合成，特别是最近发现的旁路聚合酶，是决定巨大病变是否会引起突变并最终导致癌症的关键因素。然而，这些生物学上重要现象的分子基础仍然知之甚少。本项目的主要目的是阐明（1）人类DNA修复酶识别和切除大体积DNA加合物（如来自B[a]P二醇环氧化物的加合物）的机制，以及（2）体外典型旁路聚合酶（Dpo 4、pol kappa和pol eta）催化的跨损伤合成中涉及的分子结构因素和机制。这些问题是解决使用定义明确的DNA序列与位点特异性纳入病变来自结合的B[a]P二醇环氧化物的腺嘌呤（dA）和鸟嘌呤（dG）的环外氨基在DNA中。具体目标一。采用可变碱基序列背景作为调节DNA局部结构特性的工具，确定导致立体化学定义的B[a]P-dG和B[a]P-dA病变的有效或无效核苷酸切除修复的DNA结构因子和加合物构象。 具体目标2。通过Y家族旁路聚合酶，确定并比较在不同碱基序列背景下大体积B[a]P-dG和B[a]P-dA病变的跨病变旁路的保真度和效率的差异和机制。现代计算和建模技术，NMR结构研究的基础上，将被用来获得洞察加合物的构象特性和特定的DNA扭曲附近的加合物，作为识别和去除病变的信号核苷酸切除修复蛋白。将采用分子动力学模拟方法来研究碱基序列和加合物立体化学依赖的translesion旁路，其中病变位于已知调节translesion合成的不同碱基序列背景中。

DESCRIPTION (provided by applicant): The human population is exposed to a variety of environmental cancer-causing pollutants that include fossil fuel combustion products and substances in cigarette smoke. The bay region benzo[a]pyrene (B[a]P) is the best known representative of a class of potentially carcinogenic compounds, the polycyclic aromatic hydrocarbons (PAH). Like other PAH substances, B[a]P is metabolically activated to highly reactive and mutagenic B[a]P dial epoxides that react with DNA forming adducts. Inefficient DNA repair and translesion synthesis of DNA adducts catalyzed by polymerases, especially the recently discovered bypass polymerases, are key factors that determine if a bulky lesion can give rise to mutations and ultimately to cancer. However, the molecular bases of these biologically important phenomena are still poorly understood. The major objectives of this project are to elucidate (1) the mechanisms by which human DNA repair enzymes recognize and excise bulky DNA adducts such as those derived from B[a]P diol epoxides, and (2) the molecular-structural factors and mechanisms involved in translesion synthesis catalyzed by representative bypass polymerases (Dpo4, pol kappa, and pol eta) in vitro. These questions are addressed using well defined DNA sequences with site-specifically incorporated lesions derived from the binding of B[a]P diol epoxides to the exocyclic amino groups of adenine (dA) and guanine (dG) in DNA. Specific Aim I. Determine the DNA structural factors and adduct conformations that cause efficient or inefficient nucleotide excision repair of stereochemically defined B[a]P-dG and B[a]P-dA lesions employing variable base sequence context as a tool to modulate the local structural properties of the DNA. Specific Aim 2. Determine and compare the differences and mechanisms involved in the fidelity and efficiency of translesion bypass of bulky B[a]P-dG and B[a]P-dA lesions in different base sequence contexts by Y family bypass polymerases. Modern computational and modeling techniques, based on NMR structural studies, will be employed to derive insights into adduct conformational properties and specific DNA distortions in the vicinity of the adducts that serve as signals of recognition and removal of the lesions by nucleotide excision repair proteins. Molecular dynamic simulation methods will be employed to investigate base sequence and adduct stereochemistry-dependent translesion bypass with the lesions positioned in different base sequence contexts that are known to modulate translesion synthesis.