Reaction pathways & products of guanine radicals in DNA

描述：（由申请人提供）临床和实验数据表明 慢性感染和炎症会增加 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） 增强了。最近有证据表明，碳酸根阴离子可能发挥作用， 在破坏生物大分子，特别是蛋白质和 脂质，在细胞环境中受到氧化应激。不过小 已知碳酸根与DNA的相互作用中央 本项目假设碳酸根阴离子选择性地 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。我们已经证明鸟嘌呤自由基 在双链DNA中， 与各种其它自由基物质如二氧化氮自由基形成 位点特异性硝基鸟嘌呤加合物，以及 后者具体目标1是建立这些反应的途径 具有确定碱基序列和组成的寡核苷酸中的鸟嘌呤基团。 双链DNA中鸟嘌呤自由基的浓度将是 采用激光激发瞬态吸收的真实的实时监测 光谱技术。这些鸟嘌呤自由基的进一步氧化， 碳酸根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 在由动力学定义的反应时间间隔后的化学反应产物 流动淬火法由反应生成的加合物的化学性质 碳酸根的反应将使用标准化学品和 分析技术。在具体目标2中，研究了运行中鸟嘌呤的反应性， 在结构相似的寡核苷酸中的两个、三个和四个鸟嘌呤将 评估以确定它们的反应性是否被其他侧翼增强 GS的预测理论的理由，并确定是否运行的鸟嘌呤 可以构成由碳酸盐引发的氧化性DNA损伤的热点 根的在具体目标3中，研究了DNA中鸟嘌呤自由基与 将沿着碳酸盐所述的路线评估二氧化氮 在具体目标1和2中，反应产物将是 鉴定将采用光化学方法合成 位点特异性修饰的寡核苷酸， 硝基鸟嘌呤/黄嘌呤病变。在具体目标#4中， 这些硝基鸟嘌呤/黄嘌呤病变可通过选择性基底切除进行切除 修复酶及其在定点诱变中的诱变潜力 将评估在哺乳动物细胞中的实验。

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.