Damaged DNA Recognition and Structural Basis of Mutagenesis

受损DNA的结构和能量学改变了正常的细胞功能并诱导DNA修复。氧化性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，这一过程与诱变、癌症和其他与年龄相关的疾病有关。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，并以此来提高我们对DNA糖基化酶的损伤识别和DNA聚合酶催化的翻译合成的认识。项目4的具体目标包括(a)使用多维溶液态核磁共振波谱加上受限的分子动力学模拟来建立含有外环和氧化DNA损伤的DNA双链的三维结构；(b)应用特异性标记的[15N]和[13C]外环加合物来探索与翻译合成相关的动态过程，并确定病变中参与氧化损伤识别的官能团；(c)进行无限制的分子动力学模拟，阐明DNA识别病变的机制

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.