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

项目摘要 遗传完整性在维持细胞内稳态中是重要的。化学和物理 DNA的损伤是众所周知的，但对RNA的研究较少。受损碱基的错误复制 已知会导致突变、癌症和其他疾病。在这个实验室里， 二十年来，DNA聚合酶及其与受损DNA的相互作用。我们最近的研究 实验室和其他人已经表明，DNA聚合酶有时会掺入核糖核苷三磷酸 （rNTPs）。在该实验室中，至少两种人类（h）跨损伤DNA聚合酶hpol η和hpol κ， 已经显示出对DNA和RNA模板具有意想不到的活性，包括逆转录酶活性。 转录、DNA引发和转录。将系统地研究这些活动，特别是 已知几种常见的损伤在DNA中形成，并假设也存在于RNA中。是 假设这些聚合酶的特定空间和键合特征赋予这些新的性质 并且这些生物学特性在细胞中起作用。拟议研究的特点包括：（i） hpol η在转录、逆转录和DNA引发中的活性表征。稳住- 将进行状态和预稳态动力学分析以鉴定催化特异性和限速 反应步骤。这项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- 氧代G）、1，N6-乙烯基腺嘌呤和（胸苷-胸苷）环丁烷嘧啶二聚体（CPD）），包括 分析hpol η如何在RNA模板中插入正确或不正确的碱基。x射线 晶体学将用于定义有助于观察到的催化性能的hpol η的特征。 这项工作的另一个特点是分析单个RNA加合物的水平。(ii)一些类似的问题将 用hpol κ处理，已证明hpol κ具有逆转录和DNA引发作用， 活动据报道，hpol 1能够相对于未受损的DNA插入rNTPs，这是一种脱碱基突变。 位点和8-oxoG。利用三元配合物的X-射线晶体结构研究其机理 与天然的和加合的DNA模板。(iii)hpol η与RNA反应的生物学意义 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， 在生理条件下，hpol η与CPD（被认为是hpol η最天然的底物）相反 包括金属和核糖-和2 ′-脱氧核糖核苷酸的典型细胞浓度。的影响 CPD对面的核糖核苷酸将用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， 系统性自身免疫将使用XPV成纤维细胞提取物和细胞，其在以下方面不同于对照细胞： 它们是否含有hpol η。总的来说，这些研究将提供新的见解的机制， 这些重要的聚合酶的作用以及它们的生物学功能是什么。

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.