Impact of PAH-DNA Lesions on DNA Repair and Replication

描述（由申请人提供）：人类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。湾区苯并[a]芘（B[a]P）是一类潜在致癌化合物多环芳烃（PAH）中最著名的代表。与其他多环芳烃物质一样，B[a]P被代谢激活为与DNA形成加合物反应的高活性和致突变的B[a]P表壳环氧化物。聚合酶（尤其是最近发现的旁路聚合酶）催化DNA加合物的低效率DNA修复和翻译合成是决定一个大的病变是否会引起突变并最终导致癌症的关键因素。然而，这些重要的生物学现象的分子基础仍然知之甚少。该项目的主要目标是阐明(1)人类DNA修复酶识别和去除大块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)具有代表性的旁路聚合酶（Dpo4， pol kappa和pol eta）在体外催化的翻译合成中涉及的分子结构因素和机制。这些问题是通过明确定义的DNA序列来解决的，这些DNA序列是由B[a]P二醇环氧化物与DNA中腺嘌呤（dA）和鸟嘌呤（dG）的外环氨基结合而产生的位点特异性合并病变。1 .利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病变有效或无效核苷酸切除修复的DNA结构因素和加合物构象。具体目标2。确定并比较Y家族旁路聚合酶在不同碱基序列背景下对大体积B[a]P-dG和B[a]P-dA病变进行转译旁路的保真度和效率的差异和机制。基于核磁共振结构研究的现代计算和建模技术将被用于深入了解加合物的构象性质和加合物附近的特定DNA畸变，这些加合物作为核苷酸切除修复蛋白识别和去除病变的信号。分子动力学模拟方法将用于研究碱基序列和加合物立体化学依赖的平移绕道，这些病变位于已知调节平移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.