Chemistry and Biology of Oxidized Purine Lesions in DNA

描述(由申请人提供)：细胞中的活性氧物种(ROS)会导致DNA损伤，包括碱基损伤，如果不修复，这些损伤是突变的。导致突变的分子事件现在提供了氧化DNA损伤和癌症之间的直接联系。了解肿瘤发生的分子基础是预防和治疗的基础。最近在本实验室确定了结构的特殊杂环化合物5-瓜尼基海因(Gh)和螺亚氨基二海因(Sp)是鸟苷和8-氧-7，8-二氢鸟苷(OG)被各种(ROS)氧化的主要产物，包括单线态氧和过氧基。在过去的资助期间，这些实验室研究了Gh和Sp形成的化学机制，开发了生成包含这些损伤的高纯度合成寡核苷酸的方法，研究了针对损伤的核苷酸的错误插入以及碱基切除修复(BER)酶的修复，并在体内实验中观察到这两个损伤的99%的突变率。最近在修复缺陷细菌细胞中检测到Sp，增加了这些损伤与氧化损伤和疾病的相关性。下一阶段的目标是了解Gh和Sp的极高突变率和不寻常的G到C突变的分子基础。其他氧化的嘌呤，包括OG，将在适当的时候进行研究以进行比较。具体地说，这项工作将(1)使用核磁共振和X射线结晶学技术确定Gh和Sp在双链DNA中的结构，(2)用Y-家族(病变旁路)DNA聚合酶检测与病变相反的核苷酸的错误插入，并用真核聚合酶研究序列依赖的移码突变，(3)使用BER糖基酶研究Gh和Sp损伤修复的分子机制，以及(4)利用DNA质粒中的定点结合病变进行体内突变研究。所有的具体目标都需要不断改进我们的合成程序，以生成核苷标准、核苷酸三磷酸盐和寡核苷酸底物以及病变的质谱学特征。合作者将协助进行核磁共振和X射线结晶学，以及在大肠杆菌中的体内诱变研究。该实验室将继续帮助正在开发分析方法以确定Sp和Gh损伤的细胞水平的合作者。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) in the cell lead to DNA damage including base lesions that are mutagenic if unrepaired. The molecular events leading to mutagenesis now provide a direct link between oxidative DNA damage and cancer. An understanding of the molecular basis of carcinogenesis is the foundation of prevention and treatment. The unusual heterocycles 5-guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), whose structures were recently assigned in this laboratory, are major products of guanosine and 8-oxo-7,8-dihydroguanosine (OG) oxidation by a wide variety of (ROS) including singlet oxygen and peroxyl radicals. In the past funding period, these laboratories have investigated the chemical mechanism of formation of Gh and Sp, developed methods of generating highly pure synthetic oligodeoxynucleotides containing these lesions, studied misinsertion of nucleotides opposite the lesions as well as repair by base excision repair (BER) enzymes, and observed a 99 percent mutation rate of both lesions in an in vivo assay. The recent detection of Sp in repair-deficient bacterial cells increases the relevancy of these lesions to oxidative damage and disease. The aims of the next period seek to understand the molecular basis for the extremely high mutation rates and the unusual G to C mutations observed with Gh and Sp. Other oxidized purines including OG will be studied for comparison when appropriate. Specifically, this work will (1) determine the structures of Gh and Sp in duplex DNA using NMR and x-ray crystallographic techniques, (2) examine misinsertion of nucleotides opposite the lesions with Y-family (lesion bypass) DNA polymerases and investigate sequence-dependent frameshift mutagenesis with eukaryotic polymerases, (3) study the molecular mechanisms of repair of Gh and Sp lesions using BER glycosylases, and (4) study in vivo mutagenesis using site-specifically incorporated lesions in DNA plasmids. All of the specific aims require continual refinement of our synthetic procedures for generation of nucleoside standards, nucleotide triphosphates and oligonucleotide substrates as well as mass spectrometric characterization of the lesions. Collaborators will assist with NMR and x-ray crystallography, and in vivo mutagenesis studies in E. coli. This laboratory will continue to aid collaborators who are developing analytical methods to determine the cellular levels of the Sp and Gh lesions.