Sequence Effects of Arylamine-DNA Adducts: Repair and Replication

描述（由申请人提供）：我们研究计划的长期目标是阐明DNA加合物诱导化学致癌的分子机制。芳香胺是众所周知的环境致癌物。特别地，已经在各种人体组织中证实了芳胺-DNA加合物的形成，并且认为其诱导突变。我们以前已经证明，DNA中的芳胺加合物存在于三种定义明确的构象：堆叠（S），外部B型（B）和楔形（W）。构象取决于致癌物在DNA分子中的位置，并且在生理条件下类型的群体比率是序列依赖性的。在本申请中，我们假设芳胺诱导的修复和突变是构象特异性的（S，B，W）。我们提出了四个主要的目标，以帮助定义加合物的构象，并检查其特定的修复（初始损伤识别）和复制（复制叉异质性）的影响。具体而言，这些目标集中在：（1）构象特异性修复在人类核苷酸切除修复（NER）系统和长程序列效应，（2）损伤识别（蛋白质-DNA相互作用）（3）序列依赖性滑动诱导的移码突变的热力学，和（4）复制叉构象异质性和聚合酶结合。我们将不仅采用现有的动态19 F NMR/CD，EMSA和荧光光谱，而且还采用创新的基于芯片的表面等离子体共振（SPR）和差示扫描量热法（DSC）程序，创建一套强大的生物物理方法。成功完成所提出的目标将有助于我们更好地掌握蛋白质-DNA相互作用参与人类NER和跨病变合成，这对解决癌症病因的分子细节具有重要意义。这些知识也将有助于制定合理的预防和风险评估战略。 公共卫生相关性：散发性人类癌症的主要原因是环境。芳香胺是与人类癌症病因学有关的最臭名昭著的环境化学物质之一。芳香胺-DNA加合物的形成已在各种人体组织中得到证实，并被认为可诱导化学致癌作用。因此，有必要阐明这些病变是如何修复和复制在体内的原子和分子水平。产生不良后果的关键分子（加合物结构，聚合酶，修复蛋白）必须被识别，表征和理解，以制定适当的预防和风险评估策略。

DESCRIPTION (provided by applicant): The long term goal of our research program is to elucidate the molecular mechanisms of DNA adduct-induced chemical carcinogenesis. Aromatic amines are well-known environmental human carcinogens. In particular, arylamine-DNA adduct formation has been confirmed in various human tissues and is believed to induce mutation. We have previously shown that arylamine adducts in DNA exist in three well-defined conformations: stacked (S), external B-type (B), and wedge (W). The conformation depends on the location of the carcinogen moiety in the DNA molecule, and the population ratios of the types under physiological conditions are sequence-dependent. In this application, we hypothesize that arylamine-induced repair and mutation is conformation-specific (S, B, W). We propose four major aims to help define adduct conformation and examine their specific effects on repair (initial damage recognition) and replication (replication fork heterogeneity). Specifically, these aims focus on: (1) conformation-specific repair in a human nucleotide excision repair (NER) system and long-range sequence effects, (2) damage recognition (protein-DNA interaction) (3) the thermodynamics of sequence-dependent slippage-induced frameshift mutagenesis, and (4) replication fork conformational heterogeneity and polymerase binding. We will employ not only existing dynamic 19F NMR/CD, EMSA and fluorescence spectroscopy, but also innovative chip-based surface plasma resonance (SPR) and differential scanning calorimetric (DSC) procedures, creating a powerful suite of biophysical methodologies. Successful completion of the proposed aims will help us gain a better grasp on the protein-DNA interactions involved in human NER and trans-lesion synthesis, which have important implications for resolving the molecular details of cancer etiology. Such knowledge will also be of help in the development of sensible prevention and risk assessment strategies. PUBLIC HEALTH RELEVANCE: The primary causes of sporadic human cancers are environmental. Aromatic amines are among the most notorious environmental chemicals that are implicated in the etiology of human cancers. Formation of arylamine-DNA adducts has been confirmed in various human tissues and is believed to induce chemical carcinogenesis. Hence it is imperative to elucidate how these lesions are repaired and replicated in vivo at the atomic and molecular levels. The key molecular players (adduct structures, polymerases, repair proteins) producing adverse outcomes must be identified, characterized, and understood in order to devise appropriate prevention and risk assessment strategies.