OXIDATIVE DNA DAMAGE AND THE ANALYSIS OF 8-OXOG REPAIR

DNA 氧化损伤和 8-OXOG 修复分析

• 批准号: 6043481
• 负责人: Walter Andy Deutsch
• 金额: $ 23.08万
• 依托单位: LSU PENNINGTON BIOMEDICAL RESEARCH CTR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6043481
• 关键词: DNA damage; DNA repair; Drosophilidae; alleles; enzyme activity; free radical oxygen; gel mobility shift assay; gene expression; genetic promoter element; glycosyltransferase; guanine analog; ionizing radiation; luciferin monooxygenase; messenger RNA; mutagens; mutant; northern blottings; nucleic acid sequence; oxidative stress; polymerase chain reaction; protein structure function; radiation genetics; ribosomal proteins; site directed mutagenesis; western blottings

DESCRIPTION: Reactive oxygen species generated by ionizing radiation and normal cellular respiration damages DNA and contributes to a variety of human disorders including tumor promotion. A major product of that DNA damage is the formation of 8-oxoguanine, which is a frequently occurring, highly mutagenic form of base modification produced by oxidative stress. The repair of this lesion is therefore of the utmost importance for preserving the stability of the genome. In this regard, we have cloned a Drosophila gene S3 whose encoded product apparently repairs 8-oxoguanine. This conclusion is based upon tests utilizing 5' end-labeled DNA fragments that were specifically cleaved by S3 at sites of 8-oxoguanine as revealed on DNA sequencing gels. This and other results support the notion that S3 contains N-glycosylase activity for the removal of 8-oxoguanine, although this has not been directly proven. To establish the nature of 8-oxoguanine removal by S3, defined DNA substrates that allow for the monitoring of N-glycosylase activity will be utilized. Other tests are described that should identify the types of nuclease activities associated with S3 when presented with a variety of modified 3' and 5' termini. Beyond a detailed biochemical characterization of S3, this proposal also describes experiments that will attempt to reconcile the rather bewildering array of roles apparently carried out by S3. For example, S3 has been shown to be a ribosomal protein with functions in protein synthesis. Notably, it is also a member of 6 ribosomal genes whose mRNA s are elevated in colorectal cancers. As a means of solving this conundrum, the entire gene and associated promoter will be cloned, its organization evaluated, and regulation in response to a variety of DNA damaging agents assessed. Lastly, S3 has been mapped to a region on the third chromosome that encodes a lethal Minute mutation. There are four alleles at this locus, all of which will be sequenced from heterozygotes to determine the DNA alteration. Along with some predictions from a yeast nuclease gene whose encoded product contains a domain similar to S3, we will begin to break down the S3 gene sequence by deletion mutagenesis to determine which areas are vital for its role as a DNA repair protein. Taken together, these experiments are aimed at determining the various domains utilized by a multifunctional protein to carry out its individual roles that perhaps will lead to an increased understanding of the phenotypic variability of human diseases with compromised systems of DNA repair.

描述：电离辐射和电离辐射产生的活性氧物种 正常的细胞呼吸会损伤DNA，并导致多种 包括肿瘤促进在内的人类疾病。这种DNA的一个主要产物 损伤是8-氧鸟嘌呤的形成，这是一种经常发生的， 由氧化应激产生的高度突变形式的碱基修饰。 因此，修复这一损伤对于 保持基因组的稳定性。在这方面，我们克隆了一个 果蝇基因S3，其编码产物明显修复8-氧鸟嘌呤。 这一结论是基于利用5‘端标记的DNA片段进行的测试 如上所示，在8-氧鸟嘌呤位置被S3特异性切割 DNA测序凝胶。这一结果和其他结果支持S3 含有脱除8-氧鸟嘌呤的N-糖基酶活性，尽管 这一点还没有得到直接证明。确定8-氧鸟嘌呤的性质 由S3去除，定义的DNA底物允许监测 将利用N-糖基酶活性。其他测试被描述为 应确定与S3相关的核酸酶活性的类型 呈现了各种修饰的3‘和5’末端。除了详细的 S3的生化特性，本建议还描述了实验 这将试图协调相当令人困惑的一系列角色 显然是由S3实施的。例如，S3已被证明是一个 具有蛋白质合成功能的核糖体蛋白。值得注意的是，它也是 S基因在结直肠癌中表达升高的6个核糖体基因中的一个成员 癌症。作为解决这个难题的一种手段，整个基因和 将克隆相关启动子，对其组织进行评估，并 对各种DNA损伤剂进行评估后的监管。 最后，S3已被定位到第三条染色体上的编码区域 一种致命的微小突变。在这个座位上有四个等位基因，都是 将从杂合子中对其进行测序，以确定DNA改变。 以及来自酵母核酸酶基因的一些预测，该基因的编码产物 包含一个类似于S3的结构域，我们将开始分解S3基因 通过缺失突变来确定哪些区域对ITS至关重要 作为DNA修复蛋白的作用。总而言之，这些实验的目的是 在确定多功能蛋白质所利用的各种结构域时 履行其各自的角色，这可能会导致 对人类疾病表型变异性的理解 受损的DNA修复系统。