Mechanisms of Copying of Carcinogen-damaged DNA and RNA by Translesion Polymerases

跨损伤聚合酶复制致癌物损伤的 DNA 和 RNA 的机制

基本信息

批准号：
9886242
负责人：
F PETER Guengerich
金额：
$ 35.48万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9886242
关键词：
8-hydroxyguanosine Active Sites Address Adenine Base Pairing Binding Biochemical Biological Biological Assay Biological Process Bypass Cancer Etiology Carcinogens Catalysis Cell Extracts Cell Maintenance Cells Chemicals Collaborations Complex Crystallization DNA DNA Adducts DNA Damage DNA Modification Process DNA Primers DNA biosynthesis DNA-Directed DNA Polymerase Disease Enzymes Fibroblasts Genetic Genetic Materials Genetic Transcription Goals Hela Cells High Pressure Liquid Chromatography Homeostasis Human Human Activities Individual Investigation Ions Kinetics Laboratories Lead Lesion Link Maintenance Malignant Neoplasms Metals Molecular Mutation Nucleotide Excision Repair Nucleotides Oligonucleotides Physiological Polymerase Property Pyrimidine Dimers RNA RNA primers RNA-Directed DNA Polymerase Reaction Reporting Reverse Transcription Ribonucleases Ribonucleosides Ribonucleotides Roentgen Rays Series Site Specificity Structure System Testing Thymidine Tissues Titrations Tyrosine Work X-Ray Crystallography adduct base chemical carcinogenesis experimental study human DNA in vitro activity insight interest novel polymerization repaired screening sugar systemic autoimmunity tripolyphosphate

项目摘要

PROJECT SUMMARY Genetic integrity is important in the maintenance of cellular homeostasis. Chemical and physical damage is well-known with DNA but less extensively studied with RNA. Miscopying of damaged bases is known to contribute to mutations and to cancer and other diseases. A focus in this laboratory for more than two decades has been DNA polymerases and their interactions with damaged DNA. Recent studies in our lab and by others have shown that DNA polymerases sometimes incorporate ribonucleoside triphosphates (rNTPs). In this laboratory, at least two of the human (h) translesion DNA polymerases, hpol η and hpol κ, have been shown to have unexpected activities with both DNA and RNA templates, including reverse transcription, DNA priming, and transcription. These activities will be studied systematically, particularly with several common lesions known to be formed in DNA and hypothesized to also be present in RNA. It is hypothesized that specific steric and bonding features of these polymerases impart these novel properties and that these biological properties are operative in cells. Features of the proposed studies include: (i) Characterization of the activity of hpol η in transcription, reverse transcription, and DNA priming. Steady- state and pre-steady-state kinetic analysis will be done to identify catalytic specificity and rate-limiting reaction steps. A key feature of the work will be several adducts in DNA and RNA (7,8-dihydro-8-oxoG (8- oxoG), 1,N6-ethenoadenine, and (thymidine-thymidine) cyclobutane pyrimidine dimer (CPD)), including analysis of how hpol η inserts correct or incorrect bases opposite each of these in RNA templates. X-Ray crystallography will be used to define features of hpol η contributing to the observed catalytic properties. Another feature of the work is analysis of levels of individual RNA adducts. (ii) Some similar questions will be addressed with hpol κ, which has been demonstrated to have reverse transcription and DNA priming activities. hpol ι has been reported to be capable of inserting rNTPs opposite undamaged DNA, an abasic site, and 8-oxoG. The mechanism will be investigated using X-ray crystal structures of ternary complexes with native and adducted DNA templates. (iii) The biological relevance of the hpol η reactions with RNA templates and insertion of ribonucleotides into DNA will be tested by investigating ribonucleotide insertion by hpol η opposite CPD (considered the most natural substrate for hpol η) under physiological conditions including typical cellular concentrations of metals and ribo- and 2ʹ′-deoxyribo-nucleotides. The effect of ribonucleotides opposite CPD will be examined with RNase H2 and nucleotide excision repair systems, in that CPD levels have been related to the presence of ribonucleotides in DNA, which in term are linked to systemic autoimmunity. XPV fibroblast cell extracts and cells will be utilized, which differ from control cells in whether they contain hpol η. Collectively these studies will provide new insight into the mechanisms of how these important polymerases act and what their biological functions are.

项目概要遗传完整性对于维持细胞的化学和物理平衡很重要。 DNA 损伤是众所周知的，但对 RNA 损伤碱基的错误复制研究较少。已知会导致突变、癌症和其他疾病。我们最近对 DNA 聚合酶及其与受损 DNA 的相互作用进行了二十年的研究。实验室和其他人已经证明 DNA 聚合酶有时会结合核糖核苷三磷酸 (rNTP) 在该实验室中，至少有两种人类 (h) 跨损伤 DNA 聚合酶 hpol η 和 hpol κ，已被证明对 DNA 和 RNA 模板具有意想不到的活性，包括反向我们将特别系统地研究转录、DNA 引发和转录。已知有几种常见损伤是在 DNA 中形成的，并且标记也存在于 RNA 中。保留了这些聚合酶的特定空间和键合特征赋予这些新特性这些生物学特性在细胞中发挥作用，拟议研究的特点包括：（i） hpol η 在转录、逆转录和 DNA 启动中的活性表征。将进行状态和前稳态动力学分析，以确定催化特异性和速率限制该工作的一个关键特征是 DNA 和 RNA 中的几种加合物 (7,8-二氢-8-oxoG (8- oxoG)、1,N6-乙烯腺嘌呤和(胸苷-胸苷)环丁烷嘧啶二聚体(CPD))，包括分析 hpol η 如何在 RNA 模板中的每个碱基对面插入正确或错误的碱基。晶体学将用于定义 hpol η 的特征，有助于观察到的催化性能。这项工作的另一个特点是分析单个 RNA 加合物的水平 (ii) 一些类似的问题。可以用 hpol κ 来解决，它已被证明具有逆转录和 DNA 引发作用据报道，hpol ι 能够将 rNTP 插入到未受损的 DNA（一种脱碱基）对面。位点和 8-oxoG 将使用三元配合物的 X 射线晶体结构来研究其机制。 (iii) hpol η 反应与 RNA 的生物学相关性模板和核糖核苷酸插入 DNA 将通过研究核糖核苷酸插入来测试 hpol η 在生理条件下与 CPD 相反（被认为是 hpol η 最天然的底物）包括金属和核糖核苷酸和 2′'-脱氧核糖核苷酸的典型细胞浓度的影响。与 CPD 相反的核糖核苷酸将用 RNase H2 和核苷酸切除修复系统进行检查， CPD 水平与 DNA 中核糖核苷酸的存在有关，而 DNA 中的核糖核苷酸与将使用 XPV 成纤维细胞提取物和细胞，其与对照细胞的不同之处在于它们是否含有 hpol η 总的来说，这些研究将为了解其机制提供新的见解。这些重要的聚合酶的作用及其生物学功能是什么。