Synthesis, StructUre and Replication of Carcirogen-Modified Oligonucleotides

描述（由申请人提供）：DNA的共价修饰被认为是化学致癌的第一步。接触致癌物通常是环境或工作条件、饮食或吸烟的结果。近年来，许多杂环胺（HCAs）已从熟肉中分离出来；杂环芳烃在细菌实验中是高度诱变的，在啮齿类动物和非人灵长类动物中是强致癌物。人体组织暴露于饮食相关剂量后检测到HCA-DNA加合物。人类接触杂环芳烃的情况很普遍，而且通常持续一生；流行病学研究表明，食用红肉会增加患结直肠癌、乳腺癌和前列腺癌的风险。因此，许多HCAs已被美国国家毒理学计划列为“合理预期的人类致癌物”，并可能在与饮食有关的人类癌症中发挥重要作用。我们提出了一个广泛的计划，通过化学合成、结构生物学、聚合酶学和DNA修复研究来确定局部DNA序列和HCA加合物的化学性质对HCA致癌的病因学的影响。该计划的基础是PIs实验室合成含有HCAs结构上定义的C8-和N2-dG加合物的寡核苷酸的能力（目的1）。许多hca是移码突变的有效诱导剂，我们将重点研究易移码序列中的hca加合物。肿瘤抑制基因移码失活在许多人类结直肠癌中被观察到。含HCA加合物的双链DNA的构象将通过多种技术进行研究，包括多维核磁共振；hca修饰的双链将在全长补体链（Aim 2）和双碱基缺失（Aim 3）的对面进行检测。此外，我们将对hca修饰的与y型家族聚合酶Dpo4结合的双链物插入前和插入后的配合物进行晶体学分析。该程序的一个关键假设是，滑移致突变中间体的精确构象将与加合物诱导帧移的倾向相关。这将通过使用原核生物和人类y家族DNA聚合酶的hca修饰模板的体外复制研究来验证（Aim 4）。扩展产品将使用范德比尔特开发的LC-ESI-MS-MS分析进行测序。修改的DNA模板和引物设计模拟移码机制中的离散中间体也将被研究。最后，我们提出将HCA加合物的构象与修复蛋白UvrA和XPC7HR23B对病变的识别联系起来（Aim 5）。

DESCRIPTION (provided by applicant): The covalent modification of DNA is believed to be the initial step in chemical carcinogenesis. Exposure to carcinogens is often a result of environmental or work conditions, diet or smoking. In recent years, a number of heterocyclic amines (HCAs) have been isolated from cooked meats; the HCAs are highly mutagenic in bacterial assays and potent haptocarcinogens in rodents and non-human primates. HCA-DNA adducts have been detected from human tissue after exposure to dietary relevant doses. Human exposure to HCAs is widespread and usually occurs over a lifetime; epidemiological studies have linked red meat consumption with increased risk of colorectal, breast, and prostate cancers. As such, many HCAs have been classified as "reasonably anticipated to be human carcinogens" by the National Toxicology Program and are likely to play a significant role in diet related human cancers. We have proposed an extensive program that interactively uses chemical synthesis, structural biology, polymerase enzymology, and DNA repair studies to define the influence of local DNA sequence and the chemical nature of HCA-adducts on the etiology of HCA carcinogenesis. A foundation of this program is the ability of the PIs lab to synthesize oligonucleotides containing structurally defined C8- and N2-dG adducts of HCAs (Aim 1). Many HCAs are potent inducers of frameshift mutations and we will focus on HCA-adducts in frameshift prone sequences. Frameshift inactivation of tumor suppressor genes is observed in many human colorectal cancers. The conformation of duplex DNA containing HCA adducts will be studied by a variety of techniques including multi-dimensional NMR; the HCA-modified duplexes will be examined opposite dC in a full-length complement strand (Aim 2) as well as opposite a two-base deletion (Aim 3). In addition, we will pursue crystallographic analysis of pre- and post-insertion complexes of HCA-modified duplexes bound to the model Y-family polymerase Dpo4. A key hypothesis of this program is that the precise conformation of the slipped mutagenic intermediate will correlate to the adduct's propensity to induced frameshifts. This will be tested by in vitro replication studies of HCA-modified templates using prokaryotic and human Y-family DNA polymerases (Aim 4). The extension products will be sequenced using an LC-ESI-MS-MS analysis developed at Vanderbilt. Modified DNA templates and primers designed to mimic discrete intermediates in the frameshift mechanism will also be studied. Finally, we propose to correlate the conformation of the HCA adducts with lesion recognition by the repair proteins UvrA and XPC7HR23B (Aim 5). PUBLIC HEALTH RELEVANCE: A family of chemical compounds collectively referred to as heterocyclic amines (HCAs) are produced at part- per-billion levels during the cooking of all meats; the HCAs are potent carcinogens in rodents and non-human primate. Epidemiological studies have linked red-meat consumption with increased risk for colorectal, prostate and breast cancer in human populations and the HCAs may contribute to the finding. This research program uses an interdisciplinary approach to better understand the molecular details of HCA induced cancers.