Interplay between DNA repair mechanisms in human cells and extracts

项目摘要 与人类暴露于环境遗传毒性化学污染物相关的慢性炎症 与人类癌症的病因学有关慢性炎症的特点是过度生产 活性氧和氮的物种，造成损害的细胞DNA，如果不删除细胞 防御机制，可能导致突变和癌症。反应性化学物质 中间体、它们对慢性炎症的影响以及癌症的引发是当前非常感兴趣的。 DNA氧化的主要目标是鸟嘌呤，它是最容易被破坏的核碱基。DNA修复是 这是一种至关重要的细胞防御机制，在保护基因组免受 这些DNA损伤的潜在有害作用。最重要的基本机制， 来自人类基因组的氧化产生的DNA损伤形式被广泛认为是基础 切除修复（BER）系统。然而，我们最近发现另一种重要的细胞防御 机制，核苷酸切除修复（NER），通常专门在清除庞大的和DNA螺旋- 扭曲加合物，也能够去除人类细胞中许多众所周知的氧化DNA损伤 提取模型系统。在这些体外系统中，两种修复系统相互竞争， 一些已知仅为BER底物的相同DNA底物。不过有一 机制也可能阻碍另一个，目前还不知道是否或如何BER和NER途径 由于各自的蛋白质水平和对DNA损伤底物的可用性， 与细胞提取物中的明显不同。然而，对可能的合作一无所知， 这两个主要的修复途径，BER和NER，在人类细胞中的竞争。在目标1中， BER和NER途径之间的竞争方面进行了定量探讨，在控制的 细胞提取物的环境，其中临界个体BER和NER蛋白浓度可以变化 这些非大体积氧化DNA损伤对NER的易感性的结构要求将是 通过在以前的项目期间磨练的实验和分子建模方法进行检查。 初步结果表明，BER和NER通路确实在人类中相互竞争， 目的2旨在确定BER和NER之间竞争的性质， 将位点特异性修饰的寡核苷酸底物转染到具有不同基因的人成纤维细胞中 背景已经创建了一个独特的氧化DNA损伤库，这些损伤可以通过NER修复， 或者仅通过BER，或者通过BER和NER机制两者。这个位点特异性修饰的DNA库 修复底物包括8-氧代鸟嘌呤及其更深的氧化产物，如立体异构体 螺亚氨基二海因，胍基海因，硝基咪唑鸟嘌呤氧化产物，5 '，8-环-2'- 脱氧嘌呤和链内交联鸟嘌呤-胸腺嘧啶DNA损伤。

PROJECT SUMMARY Chronic inflammation associated with human exposure to environmental genotoxic chemical contaminants has been implicated in the etiology of human cancers. Chronic inflammation is characterized by an overproduction of reactive oxygen and nitrogen species that cause damage to the cellular DNA that, if not removed by cellular defense mechanisms, may lead to mutations and cancer. The connections between reactive chemical intermediates, their impact on chronic inflammation, and the initiation of cancer, are of great current interest. The primary target of oxidation in DNA is guanine, the most easily damaged nucleobase. DNA repair is a critically important cellular defense mechanisms that plays a key role in safeguarding the genome from the potential deleterious actions of these DNA lesions. The most important basic mechanism of removal of these oxidatively generated forms of DNA damage from the human genome is widely assumed to be the base excision repair (BER) system. However, we have recently discovered that another important cellular defense mechanism, nucleotide excision repair (NER), that normally specializes in the clearing of bulky and DNA helix- distorting adducts, is also capable of removing a number of well known oxidative DNA lesions in human cell extract model systems. In these in vitro systems, the two repair systems compete with one another for the some of the same DNA substrates that have been known to be BER substrates only. However, one mechanism could also hinder the other one, and it is not known whether or how the BER and NER pathways compete with one another since the respective protein levels and availabilities to the DNA lesion substrates are markedly different than in the cell extracts. However, nothing is known about the possible cooperation and competition of these two major repair pathways, BER and NER, in human cells. In aim 1, the mechanistic aspects of the competition between the BER and NER pathways is quantitatively explored in the controlled environment of cell extracts in which the critical individual BER and NER protein concentrations can be varied at will; the structural requirements for susceptibility of these non-bulky oxidative DNA lesions to NER will be examined by experimental and molecular modeling approaches honed during previous project periods. Preliminary results indicate that the BER and NER pathways do indeed compete with one another in human fibroblasts, and aim 2 is designed to determine the nature of the competition between BER and NER following transfection of site-specifically modified oligonucleotide substrates into human fibroblasts with different genetic backgrounds. A unique library of oxidative DNA lesions has been created that are either repaired by NER only, or by BER only, or by both BER and NER mechanisms. This library of site-specifically modified DNA repair substrates includes 8-oxoguanine, and its deeper oxidation products such as the stereoisomeric spiroimininodihdantoins, guanidinohydantoin, a nitro-imidazole guanine oxidation product, 5’,8-cyclo-2’- deoxypurines, and intrastrand cross-linked guanine-thymine DNA lesions.