Damaged DNA Recognition and Structural Basis of Mutagenesis

受损DNA的结构和能量改变正常细胞功能并诱导DNA修复。氧化性DNA损伤和环外DNA损伤的存在导致DNA复制过程中的错误编码，这是一个与诱变、癌症和其他年龄相关疾病相关的过程。诱变中间体的构象，以及DNA聚合酶的性质，决定了这一过程的最终结果。这个项目的目标是进行病变的DNA的结构表征，并在这样做，推进我们的知识损伤识别的DNA糖基化酶和DNA聚合酶催化的translesion合成。项目4的具体目标包括：（a）使用多维溶液状态NMR光谱学，结合约束分子动力学模拟，建立含有环外和氧化DNA损伤的DNA双链体的三维结构;（B）应用特异性标记的[15 N]和[13 C]环外加合物，以探索与跨损伤合成相关的动态过程，并鉴定损伤中参与识别氧化损伤的官能团;（c）进行不受限制的分子动力学模拟，以阐明DNA识别病变的机制 糖基化酶和DNA双链体的受损碱基翻转的能量学。我们的结构研究旨在在分子水平上产生与结构相关的信息。 在识别受损的DNA的决定因素，并告知translesion合成事件的分析，补充诱变，结晶学和热力学研究在这个程序中进行。这些相互作用有望为损伤形成、DNA修复和诱变相关的分子机制提供新的见解。

The structure and energetics of damaged DNA alter normal cellular functions and induce DNA repair. The presence of oxidative DNA damage and exocyclic DNA lesions leads to miscoding during DNA replication, a process associated with mutagenesis, cancer and other age-related diseases. The conformation of mutagenic intermediates, together with the properties of DNA polymerases, determines the ultimate outcome of this process. The goal of this project is to perform the structural characterization of lesion-containing DNA and, in so doing, advance our knowledge of damage recognition by DNA giycosylases and of translesion synthesis catalyzed by DNA polymerases. The specific aims of Project 4 involve (a) the use of multidimensional solution-state NMR spectroscopy coupled with restrained molecular dynamics simulations to establish three-dimensional structures of DNA duplexes containing exocyclic and oxidative DNA lesions; (b) the application of specifically labeled [15N] and [13C] exocyclic adducts to explore dynamic processes related to translesion synthesis and to identify functional groups in the lesion involved in the recognition of oxidative damage; (c) the performance of unrestrained molecular dynamics simulations to elucidate mechanisms of lesion recognition by DNA glycosylases and the energetics of damaged-base eversion from DNA duplexes. Our structural studies are designed to generate information at the molecular level with respect to structural determinants in the recognition of damaged DNA, and to inform the analysis of translesion synthesis events, complementing mutagenesis, crystallographic, and thermodynamic studies conducted in this Program. These interactions are expected to provide new insights on molecular mechanisms associated with lesion formation, DNA repair, and mutagenesis.