Oxidative pathways of guanine in DNA

描述（由申请人提供）：由多种化学、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活性氮（分别为ROS和RNS）的增加导致氧化应激，导致氧化DNA损伤和DNA修复之间的不平衡。氧化产生的DNA损伤的积累增加了细胞的致突变负担，从而导致癌症。炎症部位过量产生的ROS和RNS的主要目标是鸟嘌呤，这是最容易氧化的天然核酸碱基。初级氧化步骤产生鸟嘌呤自由基，鸟嘌呤自由基与细胞亲核试剂和其他物质发生级联化学反应，导致DNA中形成各种不稳定中间体和稳定的遗传毒性鸟嘌呤病变。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）机制修复这些病变。主要工具有动态激光瞬态吸收光谱实时监测不稳定鸟嘌呤自由基和其他中间体的反应，HPLC、LC-MS/MS和MALDI-TOF/MS以及1D和2D NMR等方法分离和鉴定稳定的DNA损伤。主要发现有：(1)鸟嘌呤的单电子氧化依赖于碱基序列，(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、螺酰亚胺二乙酰胆碱（Sp）和其他损伤形成竞争，(3)G*-T*和Sp损伤是BER和NER修复机制的底物。新的具体目标建立在这些初步发现的基础上，目的是阐明鸟嘌呤自由基的竞争反应途径、稳定鸟嘌呤病变的形成以及后者通过BER和NER机制修复的易感性之间的关系。该项目的具体目的是：1)确定碱基序列对炎症性ROS和RNS（过氧亚硝酸盐、二氧化氮、超氧自由基）产生的鸟嘌呤病变分布的影响；2)比较裸DNA和核体DNA中这些病变的碱基序列依赖性形成；3)利用裸DNA和核小体DNA中的单个氧化鸟嘌呤损伤，确定对BER和NER修复机制的敏感性，并在选择的修复能力强和修复缺陷的细胞系中监测氧化鸟嘌呤损伤和修复。更好地了解模拟氧化应激条件下的DNA损伤和修复，将为发现疾病预防和临床治疗的新策略提供合理的基础。

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.