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

项目总结 遗传完整性对于维持细胞内环境的稳定很重要。化学和物理 DNA的损伤是众所周知的，但对RNA的研究较少。错误复制损坏的碱基是 已知会导致突变、癌症和其他疾病。这个实验室的关注点超过了 DNA聚合酶及其与受损DNA的相互作用已有二十年的历史。最近在我们的研究中 Lab和其他人的研究表明，DNA聚合酶有时会结合核糖核苷三磷酸 (Rntps)。在本实验室中，至少两种人(H)跨损伤dna聚合酶，hpol1η和hpol1κ， 已经被证明对DNA和RNA模板都有意想不到的活动，包括反向 转录、DNA启动和转录。将对这些活动进行系统的研究，特别是 已知在DNA中形成了几种常见的损伤，并假设也存在于RNA中。它是 假设这些聚合酶的特定空间和成键特征赋予了这些新的性质 这些生物学特性可以在细胞中发挥作用。建议研究的特色包括：(I) HpolDNA在转录、逆转录和η启动过程中的活性表征。稳定- 将进行稳态和稳态前的动力学分析，以确定催化的特异性和限速 反应步骤。这项工作的一个关键特征将是DNA和RNA中的几个加合物(7，8-二氢-8-氧-G(8-二氢-8-氧-G(8- OxoG)、1，N6-乙烯腺嘌呤和(胸苷-胸苷)环丁烷嘧啶二聚体)，包括 分析hpolRNA如何将正确或错误的碱基插入到η模板中与这些碱基相对的位置。X射线 结晶学将被用来确定有助于观察到的催化性能的hpolLη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hpolκ处理，已被证明具有逆转录和dna启动作用。 活动。据报道，hpoldna能够插入rntp，与未损伤的ι相对，这是一种基本的 Site和8-oxoG。将利用三元络合物的X射线晶体结构来研究其作用机理 带有天然和加合的DNA模板。(3)hpolRNA与η反应的生物学相关性 模板和将核糖核苷酸插入DNA的测试将通过调查核糖核苷酸插入进行 在生理条件下，hpollη与cpd(被认为是hpollη最天然的底物)相反。 包括金属和核糖核酸和2ʹ‘-脱氧核苷酸的典型细胞浓度。的影响 与CPD相反的核苷酸将用RNaseH2和核苷酸切除修复系统进行检查，在 CPD水平与DNA中核糖核苷酸的存在有关，从术语上讲，核糖核苷酸与 全身性自身免疫。将利用XPV成纤维细胞提取物和细胞，这与对照细胞在 它们是否含有HPOLη。总而言之，这些研究将为我们提供新的洞察，了解 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.