Oxidative pathways of guanine in DNA

描述(申请人提供)：由各种化学、物理和感染因素引起的慢性炎症增加了恶性细胞转化和人类癌症进展的风险。炎症细胞中活性氧和氮物种(ROS和RNS)的产生增加导致氧化应激，从而导致DNA氧化损伤和DNA修复之间的失衡。氧化产生的DNA损伤的积累增加了细胞的突变负担，从而可能导致癌症。在炎症部位过度产生的ROS和RNS的主要靶标是鸟嘌呤，这是最容易被氧化的天然核酸碱基。初级氧化步骤产生鸟嘌呤自由基，这些自由基与细胞亲核剂和其他物质发生一系列化学反应，导致DNA中各种不稳定的中间体和稳定的遗传毒性鸟嘌呤损伤的形成。在之前的项目期间，我们开发了新的方法和途径来研究鸟嘌呤自由基的反应途径，各种稳定最终产物的形成，以及通过碱基切除修复(BER)和核苷酸切除修复(NER)机制修复这些损伤。主要工具包括用动力学激光瞬时吸收光谱实时监测不稳定的鸟嘌呤自由基和其他中间体的反应，通过高效液相、LC-MS/MS和MALDI-TOF/MS分离和鉴定稳定的DNA损伤，以及一维和二维核磁共振方法。主要发现是(1)鸟嘌呤的单电子氧化是碱基序列依赖的，(2)发现了一种新的鸟嘌呤-胸腺嘧啶(G*-T*)链内交联损伤，它与8-oxoG、螺亚胺二乙内酯(Sp)和其他损伤的形成竞争，(3)G*-T*和Sp损伤是BER和NER修复机制的底物。新的特异性目标建立在这些初步发现的基础上，目的是澄清鸟嘌呤自由基的竞争反应途径、由此形成的稳定鸟嘌呤损伤与后者通过BER和NER机制修复的敏感性之间的关系。该项目的具体目标是：1)确定碱基序列对炎症性ROS和RNS(过氧亚硝酸盐、二氧化氮、超氧阴离子自由基)产生的鸟嘌呤损伤的分布的影响；2)比较这些损伤在裸DNA和核小体DNA中依赖碱基序列的形成；3)确定BER和NER修复机制的易感性，在裸DNA和核小体DNA中使用单一的氧化鸟嘌呤损伤修复机制，并监测选定的修复熟练和缺陷细胞系中的鸟嘌呤氧化损伤和修复。更好地了解模拟氧化应激条件下的DNA损伤和修复应该为发现疾病预防和临床治疗的新策略提供合理的基础。 公共卫生相关性：环境污染物、烟草烟雾、病毒感染、电离辐射和紫外线辐射以及其他外部因素导致的慢性炎症与许多人类癌症的病因密切相关。更好地了解炎症条件下的DNA损伤和DNA修复机制，将为发现新的氧化应激特异性生物标志物，开发新的炎症性疾病预防和临床治疗策略，以及炎症条件下癌症的进展提供合理的基础。

DESCRIPTION (provided by applicant): Chronic inflammation caused by diverse chemical, physical and infectious factors increases the risk of malignant cell transformations and the progression of human cancers. The enhanced production of reactive oxygen and nitrogen species (ROS and RNS, respectively) in inflammatory cells leads to oxidative stress that induces an imbalance between oxidative DNA damage and DNA repair. The accumulation of oxidatively generated DNA lesions enhances the mutagenic burden of the cells that can lead to cancer. A primary target of ROS and RNS overproduced at sites of inflammation is guanine, the most easily oxidizable natural nucleic acid base. The primary oxidation step generates guanine radicals that undergo a cascade of chemical reactions with cellular nucleophiles and other substances that lead to the formation of a variety of unstable intermediates and stable genotoxic guanine lesions in DNA. During the previous project period, we have developed new methods and approaches for investigating reaction pathways of guanine radicals, the formation of a variety of stable end-products, and the repair of these lesions by base excision repair (BER) and nucleotide excision repair (NER) mechanisms. The major tools include real time monitoring of the reactions of unstable guanine radical and other intermediates by kinetic laser transient absorption spectroscopy, the isolation and identification of the stable DNA lesions formed by HPLC, LC-MS/MS and MALDI-TOF/MS, and 1D and 2D NMR methods. Major findings are (1) that the one-electron oxidation of guanine is base-sequence dependent, (2) the discovery of a novel guanine-thymine (G*-T*) intrastrand cross-linked lesion that competes with the formation of 8-oxoG, spiroiminodihydantoins (Sp), and other lesions, and (3) that the G*-T* and Sp lesions are substrates for both BER and NER repair mechanisms. The new specific aims build on these preliminary findings with the objectives of clarifying the relationships between the competitive reaction pathways of guanine radicals, the resulting formation of stable guanine lesions, and the susceptibilities of the latter to repair by BER and NER mechanisms. The specific aims of this project are: 1) determine the effects of base sequence on the distributions of the guanine lesions generated by inflammatory ROS and RNS (peroxynitrite, nitrogen dioxide, superoxide radicals); 2) Compare the base sequence-dependent formation of these lesions in naked DNA and nucleosomal DNA; 3) Determine the susceptibilities to BER and NER mechanisms of repair using single oxidative guanine lesions in naked and nucleosomal DNA, and monitor oxidative guanine damage and repair in selected repair-proficient and deficient cell lines. A better understanding of DNA damage and repair under conditions simulating oxidative stress should provide a rational basis for discovering new strategies for the prevention and clinical treatments of disease. PUBLIC HEALTH RELEVANCE: Chronic inflammation developed in response to environmental pollutants, tobacco smoke, viral infections, ionizing and UV radiations, and other exogenous factors, is tightly implicated in the etiology of many human cancers. A better understanding of the DNA damage and DNA repair mechanisms under inflammatory conditions will provide a rational basis for discovering new specific biomarkers of oxidative stress, the development of new strategies for the prevention and clinical treatment of inflammatory diseases, and the progression of cancers under inflammatory conditions.