Chemistry and Biology of Oxidized Purine Lesions in DNA

描述（由申请方提供）：细胞中的活性氧（ROS）导致DNA损伤，包括如果未修复则具有致突变性的碱基损伤。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。了解癌症发生的分子基础是预防和治疗的基础。杂环化合物5-胍基乙内酰脲（Gh）和螺亚氨基二乙内酰脲（Sp）是鸟苷和8-氧代-7，8-二氢鸟苷（OG）被多种活性氧（包括单线态氧和过氧自由基）氧化的主要产物，其结构最近在本实验室被指定。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，开发了产生含有这些病变的高纯度合成寡脱氧核苷酸的方法，研究了病变对面核苷酸的错误插入以及碱基切除修复（BER）酶的修复，并在体内试验中观察到两种病变的99%突变率。最近在修复缺陷的细菌细胞中检测到Sp增加了这些病变与氧化损伤和疾病的相关性。下一阶段的目标是寻求了解极高的突变率和不寻常的G到C突变观察到的Gh和SP的分子基础。其他氧化嘌呤，包括OG将进行研究，以进行比较，在适当的时候。具体而言，本工作将（1）利用NMR和X-射线晶体学技术确定双链DNA中Gh和Sp的结构，（2）利用Y-家族检测损伤相对的核苷酸错误插入（损伤旁路）DNA聚合酶，并研究真核聚合酶的序列依赖性移码突变;（3）BER糖基化酶修复Gh和Sp损伤的分子机制，和（4）使用DNA质粒中的位点特异性掺入损伤研究体内诱变。所有的具体目标都需要不断完善我们的合成程序，以生成核苷标准品、三磷酸核苷和寡核苷酸底物，以及病变的质谱表征。合作者将协助核磁共振和X射线晶体学，并在E。杆菌该实验室将继续帮助合作者开发分析方法，以确定Sp和Gh病变的细胞水平。

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.