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

• 批准号: 7781467
• 负责人: Carmelo J Rizzo
• 金额: $ 34.88万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 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/7781467
• 关键词: 2-aminofluorene; Ames Assay; Amino Acids; Aromatic Amines; Binding; Biochemical; Biological; Biological Assay; Biological Process; Breast; Bypass; Calculi; Carcinogens; Chemicals; Colorectal; 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; Prostate; 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; anticancer research; 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

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(申请人提供)：DNA的共价修饰被认为是化学致癌的第一步。接触致癌物通常是由于环境或工作条件、饮食或吸烟造成的。近年来，从熟肉中分离出了许多杂环胺，这些杂环胺在细菌检测中具有高度的致突变性，在啮齿动物和非人类灵长类动物中具有强烈的触发癌作用。在暴露于饮食相关剂量后，已从人体组织中检测到HCA-DNA加合物。人类对六氯环己烷的暴露很普遍，通常发生在一生中；流行病学研究已将食用红肉与结直肠癌、乳腺癌和前列腺癌的风险增加联系起来。因此，许多六氯环己烷已被国家毒理学计划归类为“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，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癌变的影响。这一计划的基础是PIS实验室合成含有结构定义的HCA的C8和N2-DG加合物的寡核苷酸的能力(目标1)。许多HCA是移码突变的有效诱导者，我们将重点关注移码序列中的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的构象将被包括多维核磁共振在内的各种技术研究；HCA修饰的双链将在全长补体链中与DC相对(目标2)以及与两个碱基缺失相反(目标3)。此外，我们将对与模型Y-家族聚合酶Dpo4结合的HCA修饰的双链插入前和插入后的复合体进行结晶学分析。这个计划的一个关键假设是，滑动的诱变中间体的精确构象将与加合物诱导框架移位的倾向相关。这将通过使用原核生物和人类Y-家族DNA聚合酶(AIM 4)对HCA修饰的模板进行体外复制研究来验证。扩展产品将使用Vanderbilt开发的LC-ESI-MS-MS分析进行测序。为模拟移码机制中的离散中间体而设计的修改的DNA模板和引物也将被研究。最后，我们建议将HCA加合物的构象与修复蛋白UvrA和XPC7HR23B(目标5)的病变识别相关。 与公共健康相关：一类统称为杂环胺(HCAs)的化合物在所有肉类的烹调过程中以十亿分之一的水平产生；HCAs对啮齿动物和非人类灵长类动物是强有力的致癌物质。流行病学研究已经将食用红肉与人类患结直肠癌、前列腺癌和乳腺癌的风险增加联系在一起，而HCA可能是这一发现的原因之一。这项研究计划使用跨学科的方法来更好地了解HCA诱导的癌症的分子细节。