Sequence Effects of Arylamine-DNA Adducts: Repair and Replication

项目总结/摘要 我们研究计划的长期目标是阐明DNA的分子机制 内收诱导的化学致癌作用。芳香胺是众所周知的环境人类 致癌物质。特别地，已经在各种人类中证实了芳基胺-DNA加合物的形成。 组织，并被认为会诱发突变。我们以前已经表明，芳胺加合物在DNA 存在三种明确定义的构象：堆叠（S）、外部B型（B）和楔形（W）。的 构象取决于致癌物部分在DNA分子中的位置， 在生理条件下，各类型的种群比例是序列依赖性的。在这 应用，我们假设芳胺诱导的修复和突变是构象（S/B/W） 特定.我们提出了四个主要目标，以帮助定义加合物构象，并检查其具体的 对修复（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，（2）长程序列效应，（3）序列依赖的热力学 滑动诱导移码突变，和（4）复制叉构象 异质性我们将不仅采用现有的动态19 F NMR/CD，EMSA和荧光 光谱，而且创新的基于芯片的表面等离子体共振（SPR）和差分 扫描量热法（DSC）程序，创造了一个强大的生物物理方法套件。 这些目标的成功实现将有助于我们更好地理解蛋白质-DNA 参与人类NER和跨病变合成的相互作用，这对 解决癌症病因的分子细节。这样的知识也将有助于 制定合理的预防和风险评估战略。

Project Summary/Abstract 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 (S/B/W) specific. 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, (2) long-range sequence effects, (3) the thermodynamics of sequencedependent slippage-induced frameshift mutagenesis, and (4) replication fork conformational heterogeneity. 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.