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、衰老和神经问题 精神错乱。8-氧代-7，8-二氢鸟苷(OG)被认为是最多的 由氧化DNA损伤引起的严重损伤，由于其能力 与A错配，如果不修复，会导致G-*T颠换突变。 这个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的单电子氧化现已在本实验室建立为 双链DNA和核苷中的螺亚氨基二海因(Sp)衍生物，而 双链DNA的主要产物是两种异构体的混合物，一种 鸟苷海因(Gh)和亚氨基尿囊素(Ia)类似物。这些损伤是 也是由G使用单线态氧和过氧基直接氧化形成的 激进分子。这个项目的中心假设是，这些活动 有聚合酶和DNA修复酶的病变(Sp和Gh/Ia) 由于(A)条件下OG的形成频率的进一步研究 氧化应激，(B)OG对进一步氧化的高反应性 与正常的DNA碱基相比，(C)OG作为热点的能力 对于通过双链DNA中远程电子转移而造成的进一步氧化损伤， 以及(D)最近发现的这些损害是 本项目的具体目标是：(1)研究反应条件 导致Sp、Gh和Ia体外损伤的单电子比较 氧化剂为其他氧化剂，如单线态氧、超氧化物和羟基 自由基，(2)开发合成方法，用于掺入纯净的， 将特征性良好的病变转化为寡核苷酸，(3)确定 Sp和Gh/Ia对双链低聚物结构的影响 (A)双链稳定性和(B)核磁共振，(4)检查聚合酶加工 含有这些损伤的DNA低聚物使用各种DNA和RNA 聚合酶和(5)研究这些病变的DNA修复活性 有fpg、yoggi、yogg2、hOggi和mut。方法论将严重依赖于 含有各种病变的合成低聚物，其结构分析 用LC-MS和2D-核磁共振、体外酶动力学分析和体内研究 大肠埃希菌。合作研究将包括#年对Sp和Gh/Ia的调查 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.