CHEMISTRY AND BIOLOGY OF OXIDIZED 8OXO G LESIONS IN DNA

描述：（申请人提供）氧化性DNA损伤是导致 广泛的健康问题，包括癌症，衰老和神经系统 紊乱8-氧代-7，8-二氢鸟苷（OG）被认为是可能是最 氧化性DNA损伤导致的严重损伤， 与A错配，如果未修复，则导致G-*T颠换突变。 该实验室和其他实验室最近的研究表明，OG是一种不稳定的 在各种氧化条件下的中间体。的产品 OG的单电子氧化现在已经在这个实验室中被建立为 螺旋亚氨基二乙内酰脲（Sp）衍生物在ss DNA和核苷，而 双链DNA的主要产物是两种异构体的混合物， 胍基乙内酰脲（Gh）和亚氨基尿囊素（Ia）类似物。这些病变 也可通过使用单线态氧和过氧基氧化G而直接形成 根的该项目的核心假设是，这些活动的 病变（Sp和Gh/ Ia）与聚合酶和DNA修复酶保证 进一步的研究，因为（a）在条件下形成OG的频率 （B）OG对进一步氧化的高反应性 与正常DNA碱基相比，（c）OG作为热点的能力 对于通过双链体DNA中的远程电子转移的进一步氧化损伤， 和（d）最近发现这些病变是主要的氧化产物， G.本课题的具体目标是：（1）研究反应条件 在体外通过比较单电子 氧化剂转化为其他氧化剂，如单线态氧、超氧化物和羟基 自由基，（2）开发合成方法，将纯， 将特征明确的病变转化为寡核苷酸，（3）确定 Sp和Gh/Ia对双链体寡聚体的结构影响 (a)双链体稳定性和（B）NMR，（4）检查聚合酶加工 使用各种DNA和RNA，含有这些病变的DNA寡聚体 聚合酶和（5）研究这些病变的DNA修复活性 Fpg、yOggi、yOgg2、hOggi和MutT。该方法将在很大程度上依赖于 含有各种病变的合成低聚物，它们的结构分析 通过LC-MS和2D-NMR，体外酶动力学分析和体内研究， E.杆菌合作研究将包括研究Sp和Gh/Ia， 体内诱变研究。

DESCRIPTION:(PROVIDED BY APPLICANT) Oxidative DNA damage is a contributor to a wide range of health problems including cancer, aging and neurological disorders. 8-Oxo-7,8-dihydroguanosine (OG) is regarded as perhaps the most critical lesion resulting from oxidative DNA damage due to its capability to mispair with A and, if unrepaired, to lead to a G-*T transversion mutation. Recent studies in this laboratory and others have shown that OG is an unstable intermediate under a variety of oxidative conditions. The products of one-electron oxidation of OG have now been established in this lab as a spiroiminodihydantoin (Sp) derivative in ss DNA and in nucleosides, while the major product in duplex DNA is a mixture of two isomeric species, a guanidinohydantoin (Gh) and an iminoallantoin (Ia) analog. These lesions are also formed directly by oxidation of G using singlet oxygen and peroxyl radicals. The central hypothesis of this project is that the activity of these lesions (Sp and Gh/ Ia) with polymerases and DNA repair enzymes warrants further study because of (a) the frequency of formation of OG under conditions of oxidative stress, (b) the high reactivity of OG toward further oxidation compared to the normal DNA bases, (c) the ability of OG to act as a hot spot for further oxidative damage via long-range electron transfer in duplex DNA, and (d) the recent finding that these lesions are primary oxidation products of G. The specific aims of this project are (1) to study the reaction conditions that lead to Sp, Gh and Ia lesions in vitro by comparison of one-electron oxidants to other oxidants such as singlet oxygen, superoxide, and hydroxyl radical, (2) to develop synthetic methods for incorporation of pure, well-characterized lesions into oligonucleotides, (3) to determine the structural effects of Sp and Gh/Ia on duplex oligomers through an analysis of (a) duplex stability and (b) NMR, (4) to examine the polymerase processing of DNA oligomers containing these lesions using a variety of DNA and RNA polymerases and (5) to investigate the DNA repair activity of these lesions with Fpg, yOggi, yOgg2, hOggi and MutT. The methodology will rely heavily on synthetic oligomers containing the various lesions, their structural analysis by LC-MS and by 2D-NMR, in vitro enzyme kinetic analysis and in vivo studies in E. coli. Collaborative studies will include investigations of Sp and Gh/Ia in in vivo mutagenesis studies.