Synthesis, StructUre and Replication of Carcirogen-Modified Oligonucleotides

致癌物修饰寡核苷酸的合成、结构和复制

基本信息

批准号：
8369307
负责人：
Carmelo J Rizzo
金额：
$ 34.05万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-12-15 至 2014-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8369307
关键词：
2-aminofluorene Ames Assay Amino Acids Aromatic Amines Binding Biochemical Biological Biological Assay Biological Process Bypass Calculi Carcinogens Chemicals Colorectal Cancer Complement Complex CpG dinucleotide DNA DNA Adducts DNA Binding DNA Damage DNA Modification Process DNA Sequence DNA-Directed DNA Polymerase Diet Dinucleotide Repeats Disease Domestic Fowls Dose Epidemiologic Studies Etiology Exposure to Family Fishes Foundations Frameshift Mutation Health Heterocyclic Amines Human In Vitro Laboratory Animals Lead Length Lesion Link Malignant Neoplasms Malignant neoplasm of prostate Meat Minor Modeling Molecular Molecular Conformation Mutagenesis Mutagens National Toxicology Program Nature Nucleotide Excision Repair Oligonucleotides Play Polymerase Population Positioning Attribute Predisposition Proteins Recombinants Repair Enzymology Research Resources Risk Rodent Role Site Smoking Structure Techniques Testing Time Tumor-Suppressor Gene Inactivation Tumorigenicity Work Xeroderma Pigmentosum adduct analog base carcinogenesis chemical carcinogenesis chemical property chemical synthesis citrate carrier cooking design detoxication exposed human population genotoxicity human tissue in vitro testing in vivo interdisciplinary approach interest malignant breast neoplasm nonhuman primate professor programs public health relevance red meat consumption repaired structural biology sugar tumorigenic

项目摘要

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).

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，人们从熟肉中分离出了多种杂环胺（HCA）； HCA 在细菌检测中具有高度诱变性，并且在啮齿动物和非人类灵长类动物中具有强触致癌性。在接触膳食相关剂量后，已从人体组织中检测到 HCA-DNA 加合物。人类接触 HCA 的情况很普遍，并且通常会在一生中发生；流行病学研究表明，食用红肉与结直肠癌、乳腺癌和前列腺癌的风险增加有关。因此，许多 HCA 已被国家毒理学计划归类为“合理预期为人类致癌物”，并且可能在与饮食相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，交互地使用化学合成、结构生物学、聚合酶酶学和 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 致癌病因学的影响。该计划的基础是 PI 实验室能够合成含有结构明确的 HCA C8-和 N2-dG 加合物的寡核苷酸（目标 1）。许多 HCA 是移码突变的有效诱导物，我们将重点关注移码倾向序列中的 HCA 加合物。在许多人类结直肠癌中观察到肿瘤抑制基因的移码失活。含有 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 NMR； HCA 修饰的双链体将在全长补体链中与 dC 相对的位置（目标 2）以及两碱基缺失的位置（目标 3）进行检查。此外，我们将对与模型 Y 家族聚合酶 Dpo4 结合的 HCA 修饰双链体的插入前和插入后复合物进行晶体学分析。该程序的一个关键假设是，滑动的诱变中间体的精确构象将与加合物诱导移码的倾向相关。这将通过使用原核和人类 Y 家族 DNA 聚合酶的 HCA 修饰模板的体外复制研究进行测试（目标 4）。延伸产物将使用范德堡大学开发的 LC-ESI-MS-MS 分析进行测序。还将研究旨在模拟移码机制中离散中间体的修饰 DNA 模板和引物。最后，我们建议将 HCA 加合物的构象与修复蛋白 UvrA 和 XPC7HR23B 的病变识别相关联（目标 5）。