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中产生的活性氧（ROS）对基因组稳定性构成重大威胁。由活性氧直接氧化DNA或由脂质过氧化产物修饰导致突变性DNA加合物的形成，这与致癌有关。多种DNA加合物在DNA氧化后产生，包括3-(2！-脱氧-“-d -红-戊呋喃基”-嘧啶[1,2-a]purin-10(3H)-one （M1dG）和n6 -氧丙烯- da （OPdA）。M1dG和OPdA具有独特的亲电反应性，这使它们能够与多种亲核试剂反应。OPdA是反应性较强的加合物，与N#-乙酰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机理尚不清楚，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结合蛋白来阐明交联形成的化学过程，并优化内合蛋白的检测和鉴定方法。然后，我将在哺乳动物细胞的核提取物中鉴定与OPdA加合物交联的蛋白质。尽管这些偶联物可以诱导有害的细胞效应，但DNA损伤和蛋白质之间共价交联的形成尚未得到广泛的研究。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。这项研究首次阐明了OPdA与dna结合蛋白的相互作用。

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.