Reaction pathways & products of guanine radicals in DNA

描述：(申请人提供)临床和实验数据显示 慢性感染和炎症与增加风险有关 用来发展人类癌症。为了应对炎症和感染， 细胞产生多种活性氧(ROS)和氮(RNS) 是增强的。最近有证据表明，碳酸盐自由基阴离子可能起到 在破坏生物大分子，特别是蛋白质和 脂质，在细胞环境中遭受氧化应激。然而，几乎没有 已知碳酸盐自由基与DNA的相互作用。中环 这个项目的假设是碳酸盐自由基阴离子有选择性地 通过单电子转移过程氧化鸟嘌呤，从而导致 DNA中鸟嘌呤自由基的形成。我们已经证明了鸟嘌呤自由基 在双链DNA中足够长的寿命(大约几秒钟)来做出反应 与各种其他自由基物种，如二氧化氮自由基形成 特定部位的硝基鸟嘌呤加合物，以及 后者。具体目标1是建立这些化合物的反应途径 确定碱基序列和组成的寡核苷酸中的鸟嘌呤自由基。 双链DNA中鸟嘌呤自由基的浓度将为 利用激光激发瞬时吸收进行实时监测 光谱学技术。这些鸟嘌呤自由基的进一步氧化 碳酸盐自由基及其与超氧自由基离子的反应将是 作为时间的函数进行监测，以及出现的动力学 由动力学定义的反应时间间隔后的化学反应产物 流动猝灭法。加合物的化学性质是由 碳酸盐自由基的反应将使用标准的化学和 分析技术。在具体目标2中，鸟嘌呤在奔跑中的反应性 结构相似的寡核苷酸中的两个、三个和四个鸟嘌呤将 被评估以确定它们的反应性是否被其他侧翼增强 在理论基础上预测的GS，并确定鸟嘌呤的运行 可构成碳酸盐引发DNA氧化损伤的热点 激进分子。在特定目标3中，DNA中鸟嘌呤自由基与 二氧化氮将按照碳酸盐的描述路线进行评估。 特定目标1和2中的自由基，反应产物为 已确认身份。将使用光化学方法来合成 具有明确单链的定点修饰寡核苷酸 硝基鸟嘌呤/黄嘌呤损伤。在具体目标#4中， 这些硝基鸟/黄嘌呤病变要通过选定的碱基切除来切除 定点突变中的修复酶及其诱变潜能 在哺乳动物细胞中的实验将被评估。

DESCRIPTION: (provided by applicant) Clinical and experimental data suggest that chronic infection and inflammation are associated with an increased risk for developing human cancers. In response to inflammation and infection, the cellular production of diverse reactive oxygen (ROS) and nitrogen (RNS) species is enhanced. There is recent evidence that carbonate radical anions may play an important role in damaging biological macromolecules, especially proteins and lipids, in cellular environments subjected to oxidative stress. However, little is known about the interactions of carbonate radicals with DNA. The central hypothesis of this project is that carbonate radical anions site-selectively oxidize guanines by a one-electron transfer process, thus resulting in the formation of guanine radicals in DNA. We have shown that the guanine radicals in double stranded DNA are sufficiently long-lived (about seconds) to react with various other radical species such as nitrogen dioxide radicals to form site-specific nitroguanine adducts, as well as decomposition products of the latter. Specific aim 1 is to establish the pathways of reaction of these guanine radicals in oligonucleotides of defined base sequence and composition. The concentrations of the guanine radicals in double stranded DNA will be monitored in real time employing laser excitation transient absorption spectroscopy techniques. The further oxidation of these guanine radicals by carbonate radicals and their reactions with superoxide radical ions will be monitored as a function of time, together with the kinetics of appearance of chemical reaction products after reaction time intervals defined by kinetic flow-quench methods. The chemical nature of adducts resulting from the reactions of carbonate radicals will be identified using standard chemical and analytical techniques. In specific aim 2, the reactivities of guanines in runs of two, three, and four guanines in structurally similar oligonucleotides will be assessed to determine if their reactivities are enhanced by other flanking Gs as predicted on theoretical grounds, and to determine if runs of guanines can constitute hotspots of oxidative DNA damage initiated by carbonate radicals. In specific aim 3 the reactivities of guanine radicals in DNA with nitrogen dioxide will be assessed along the lines described for the carbonate radicals in specific aims 1 and 2, and the reaction products will be identified. A photochemical method will be employed to synthesize site-specifically modified oligonucleotides with well-defined single nitroguanine/xanthine lesions. In specific aim #4, the susceptibilities of these nitroguanine/xanthine lesions to excision by selected base excision repair enzymes, and their mutagenic potentials in site-directed mutagenesis experiments in mammalian cells will be assessed.