Elucidating the Chemistry and Targets of Cross-linking by Endogenous DNA Damage

描述（由申请人提供）：细胞代谢和暴露于环境因子产生的活性氧（ROS）对基因组稳定性构成重大威胁。ROS直接氧化DNA或脂质过氧化修饰副产物导致形成与致癌有关的致突变DNA加合物。在DNA氧化后产生多种DNA加合物，包括3-（2！脱氧-“-D-吡喃-戊呋喃糖基）-嘧啶并[1，2-a]嘌呤-10（3 H）-酮（M1 dG）和N6-氧代丙烯基-dA（OPdA）。M1 dG和OPdA具有独特的亲电反应性，这使得它们能够与多种亲核试剂反应。OPdA是反应性更强的加合物，并与N#-乙酰基赖氨酸形成共价交联。这增加了OPdA与DNA结合蛋白形成交联的可能性。这种DNA-蛋白质交联形成的化学过程还没有被很好地理解，这是一个令人兴奋的新发现领域。我建议使用EcoRI作为模型DNA结合蛋白，以阐明化学交联形成和优化的方法检测和鉴定加合蛋白。然后，我将确定蛋白质交联OPdA加合物在哺乳动物细胞的核提取物。DNA损伤和蛋白质之间共价交联的形成尚未被广泛研究，尽管这些缀合物可以诱导有害的细胞效应。本提案中概述的研究将增加我们对交联如何形成以及哪些蛋白质能够与OPdA形成交联的理解。本研究首次阐明了OPdA与DNA结合蛋白的相互作用。 公共卫生相关性：活性氧的产生发生在正常的细胞功能期间，并且通过慢性炎症和暴露于环境因子而增加。DNA是活性氧攻击的目标，所产生的损伤与致癌作用有关。这些研究将扩大我们对活性氧诱导的DNA损伤如何通过检查表现出与DNA结合蛋白形成交联的能力的内源性DNA损伤来促进致癌作用的理解。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) produced from cellular metabolism and exposure to environmental agents pose a significant threat to genomic stability. Direct DNA oxidation by ROS or modification by products of lipid peroxidation result in the formation of mutagenic DNA adducts that are implicated in carcinogenesis. A variety of DNA adducts are produced following DNA oxidation including 3-(2!-deoxy- "-D-erythro-pentofuranosyl)- pyrimido[1,2-a]purin-10(3H)-one (M1dG) and N6-oxopropenyl-dA (OPdA). M1dG and OPdA possess the unique characteristic of electrophilic reactivity, which enables them to react with a variety of nucleophiles. OPdA is the more reactive adduct and forms covalent cross-links with N#-acetyl lysine. This raises the possibility that OPdA forms cross-links with DNA-binding proteins. The chemistry of this DNA-protein cross-link formation is not well understood and presents an exciting and novel area for discovery. I propose to use EcoRI as a model DNA-binding protein to elucidate the chemistry of cross-link formation and to optimize the methods for detection and identification of adducted proteins. I will then identify the proteins cross-linked to OPdA adducts in nuclear extracts of mammalian cells. The formation of covalent cross-links between DNA damage and protein has not been extensively investigated despite the detrimental cellular effects these conjugates can induce. The studies outlined in this proposal will increase our understanding of how cross-links are formed as well as what proteins are able to form cross-links with OPdA. This investigation represents the first study to elucidate the interaction of OPdA with DNA-binding proteins. PUBLIC HEALTH RELEVANCE: The production of reactive oxygen species occurs during normal cellular functions and is increased by chronic inflammation and exposure to environmental agents. DNA is a target for attack by reactive oxygen and the damage produced has been linked to carcinogenesis. These studies will expand our understanding of how DNA damage induced by reactive oxygen contributes to carcinogenesis by examining endogenous DNA lesions that exhibit the ability to form cross-links to DNA-binding proteins.