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：
8196770
负责人：
Carmelo J Rizzo
金额：
$ 34.75万
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/8196770
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). 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.

描述（由申请人提供）：DNA的共价修饰被认为是化学癌变的第一步。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，从煮熟的肉中分离出许多杂环胺（HCA）。 HCA在细菌测定中是高度诱变的，在啮齿动物和非人类灵长类动物中有效的触发性触觉。暴露于饮食相关剂量后，已经从人体组织中检测到HCA-DNA加合物。人类对HCA的暴露是普遍的，通常发生在一生中。流行病学研究已将红肉消耗与大肠癌，乳腺癌和前列腺癌的风险增加联系在一起。因此，许多HCA被国家毒理学计划归类为“合理地预期将是人类致癌物”，并且很可能在与饮食相关的人类癌症中发挥重要作用。我们提出了一项广泛的程序，该程序使用化学合成，结构生物学，聚合酶酶学和DNA修复研究来定义局部DNA序列的影响以及HCA-辅助对HCA癌发生的病因的影响。该程序的基础是PIS实验室合成含有HCAS的结构定义的C8-和N2-DG加合物的寡核苷酸（AIM 1）。许多HCA是移码突变的有效诱导剂，我们将专注于易移什式易于序列的HCA添加物。在许多人类结直肠癌中观察到肿瘤抑制基因的移灭失活。包括多维NMR在内的多种技术，将研究含有HCA加合的双链DNA的构象。 HCA修饰的双链体将在全长补体链（AIM 2）以及两层删除（AIM 3）的相对DC中进行检查。此外，我们将对与模型Y-家庭聚合酶DPO4结合的HCA修饰双链体的插入前和后插入复合物进行晶体学分析。该程序的一个关键假设是，滑倒的诱变中间体的精确构象将与加合物对诱导的移交的倾向相关。这将通过使用原核和人Y-家庭DNA聚合酶对HCA修饰模板进行体外复制研究进行测试（AIM 4）。扩展产品将使用范德比尔特（Vanderbilt）开发的LC-ESI-MS-MS分析进行测序。还将研究用于模仿移码机制中的离散中间体的修饰DNA模板和底漆。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（AIM 5）相关联。公共卫生相关性：在所有肉类烹饪过程中，共同称为杂环胺（HCA）的化合物家族统称为杂环胺（HCA）。 HCA是啮齿动物和非人类灵长类动物中有效的致癌物。流行病学研究已将红蛋糕的消费与人群中结直肠癌，前列腺癌和乳腺癌的风险增加联系起来，HCA可能有助于这一发现。该研究计划采用跨学科方法来更好地了解HCA诱导癌症的分子细节。