OXIDATIVE DNA DAMAGE AND THE ANALYSIS OF 8-OXOG REPAIR

描述：电离辐射产生的活性氧， 正常的细胞呼吸会破坏DNA，并导致多种 人类疾病，包括肿瘤促进。 DNA的主要产物 损伤是形成8-氧代鸟嘌呤，这是一种经常发生的， 由氧化应激产生高度致突变形式的碱基修饰。 因此，修复这一病变对 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，其编码的产物明显修复8-氧代鸟嘌呤。 这一结论是基于利用5'末端标记的DNA片段的测试 在8-氧代鸟嘌呤位点被S3特异性切割， DNA测序凝胶。 这一结果和其他结果支持了S3 含有去除8-氧代鸟嘌呤的N-糖基化酶活性，尽管 这一点尚未得到直接证实。 确定8-氧代鸟嘌呤的性质 通过S3去除定义的DNA底物，允许监测 将利用N-糖基化酶活性。 描述了其他测试， 应确定与S3相关的核酸酶活性类型， 呈现多种修饰的3'和5'末端。 除了详细的 S3的生化表征，该提案还描述了实验 它将试图调和那些令人困惑的角色 显然是由S3执行的。 例如，S3已被证明是一个 在蛋白质合成中起作用的核糖体蛋白质。 值得注意的是， 6个核糖体基因的成员，其mRNA在结直肠癌中升高 癌的 作为解决这个难题的一种手段，整个基因和 将克隆相关的启动子，评估其组织， 调节响应于评估的各种DNA损伤剂。 最后，S3已经被定位到第三条染色体上的一个区域，该区域编码 致命的微小突变 在这个基因座上有四个等位基因， 将从杂合子中测序以确定DNA改变。 沿着一些来自酵母核酸酶基因的预测，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它的 作为DNA修复蛋白。 综合起来，这些实验旨在 在确定多功能蛋白质利用的各种结构域时， 发挥其各自的作用，这可能会导致增加 了解人类疾病的表型变异， 受损的DNA修复系统

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.