Damaged DNA Recognition and Structural Basis of Mutagenesis

受损DNA的结构和能量改变了正常的细胞功能，并诱导DNA修复。氧化DNA损伤和外环DNA损伤的存在导致DNA复制过程中的错误编码，这一过程与突变、癌症和其他年龄相关疾病有关。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。这个项目的目标是对含有损伤的DNA进行结构表征，并在这样做的过程中，促进我们对DNA糖基酶的损伤识别和DNA聚合酶催化的跨损伤合成的了解。项目4的具体目标涉及：(A)使用多维溶液状态核磁共振波谱与受限分子动力学模拟相结合来建立含有外环和氧化DNA损伤的DNA双链的三维结构；(B)应用特定标记的[15N]和[13C]外环加合物来探索与跨损伤合成相关的动态过程，并识别参与识别氧化损伤的损伤中的官能团；(C)无限制分子动力学模拟的性能，以阐明DNA识别损伤的机制 糖基酶与dna双链受损碱基外翻的能量学。我们的结构研究是为了在分子水平上产生关于结构的信息 识别受损DNA的决定因素，并为跨损伤合成事件的分析提供信息，补充本计划中进行的诱变、结晶学和热力学研究。这些相互作用有望为与病变形成、DNA修复和突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.