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

描述(由申请人提供)：细胞新陈代谢产生的活性氧物种(ROS)和暴露在环境介质中对基因组的稳定性构成重大威胁。ROS直接氧化DNA或脂质过氧化的副产物会导致突变DNA加合物的形成，这些加合物与癌症的发生有关。DNA氧化生成多种DNA加合物，包括3-(2！-脱氧-“-D-赤基-5-呋喃葡萄糖基)-嘧啶并[1，2-a]紫红-10(~3H)-酮(M_1dG)和N_6-氧代丙烯-D(OPdA)。M1dG和OPdA具有独特的亲电反应性，使它们能够与各种亲核试剂反应。OPdA是活性较强的加合物，与N#-乙酰赖氨酸形成共价交联键。这增加了OPdA与DNA结合蛋白形成交联链的可能性。这种DNA-蛋白质交联链形成的化学机制还不是很清楚，这是一个令人兴奋的新发现领域。我建议使用EcoRI作为DNA结合蛋白的模型，以阐明交联形成的化学机制，并优化加合物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液中鉴定与OPdA加合物交叉连接的蛋白质。DNA损伤和蛋白质之间的共价交联链的形成还没有得到广泛的研究，尽管这些偶联物可以诱导有害的细胞效应。这项提案中概述的研究将增加我们对交联链是如何形成的以及哪些蛋白质能够与OPdA形成交联链的理解。这项研究是首次阐明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.