Sequence Effects of Arylamine-DNA Adducts: Repair and Replication

描述(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质。特别是，芳胺-DNA加合物的形成已经在各种人类组织中得到证实，并被认为可以诱导突变。我们以前已经证明了DNA中的芳胺加合物以三种明确的构象存在：堆积(S)、外部B型(B)和楔形(W)。构象依赖于致癌物部分在DNA分子中的位置，而生理条件下类型的种群比率是顺序依赖的。在这个应用中，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目标来帮助定义加合物构象，并研究它们对修复(初始损伤识别)和复制(复制叉异质性)的特定影响。具体地说，这些目标集中在：(1)人类核苷酸切除修复(NER)系统中的构象特异性修复和远程序列效应，(2)损伤识别(蛋白质-DNA相互作用)，(3)序列依赖滑移诱导的移码突变的热力学，以及(4)复制分叉构象异质性和聚合酶结合。我们将不仅使用现有的动态19F核磁共振/CD、EMSA和荧光光谱，而且还将使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(DSC)程序，创建一套强大的生物物理方法。这些目标的成功完成将有助于我们更好地掌握人类NER和跨病变合成中涉及的蛋白质-DNA相互作用，这对解决癌症病因学的分子细节具有重要意义。这些知识还将有助于制定合理的预防和风险评估战略。与公共卫生相关：散发性人类癌症的主要原因是环境因素。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。芳胺-DNA加合物的形成已在多种人体组织中得到证实，并被认为是诱导化学致癌的因素。因此，在原子和分子水平上阐明这些损伤是如何在体内修复和复制的势在必行。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，以便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.