BIOCHEMICAL GENETICS OF OXIDIZED DNA REPAIR IN YEAST

自发突变是导致人类遗传病的重要原因 并形成了其遗传毒性的背景 必须对环境代理人进行评判。尽管存在这些核心关切， 影响自发突变率的细胞过程是 人们对此知之甚少。该项目解决了有关 真核生物自发诱变的生化机制：IS 受基因转录和加工过程影响的自发突变 复制(目标1)？自发突变是由新陈代谢驱动的 产生DNA损伤，如氧自由基(目标2-4)？做可修复的吗 自发性DNA损伤逃避纠正并导致正常人群突变 细胞(目标3)？他们产生突变的概率更大 动力学问题(修复与复制)或损坏的速度 是生成的(目标4)？内源性DNA损伤导致的DO突变 从处理环境诱变物的主动处理途径(AIM 5)?为了达到这些目的，我们将利用我们的能力来调节 失活对酿酒酵母DNA修复能力的影响 我们之前克隆的APN1基因，它编码主要的内切酶 对于这个有机体中的基本位置。缺乏这种活性的菌株具有 显著提高的自发突变率，这是在 部分由DNA糖基酶(MAG基因产物)产生的碱性位点所致 作用于内源性DNA损伤。我们将构建测试菌株， 一个突变的目标是人类起源，与其他项目相同。 用一种新的方法快速测定突变谱 将允许我们梳理出限制正常基因的特定事件 稳定性。这些目标将以不同的方向插入 关于复制和转录(领先链相对于滞后链， 以及转录的链与非转录的链)。内源性损伤的作用 将通过删除APN1或MAG基因以及通过培养 细胞在不同的条件下(好氧与厌氧等)。酵母菌 还将构建具有更高水平的Apn1蛋白或 人类基本核酸内切酶，它将揭示是否修复 这种损伤在正常细胞中是有限的。最后，RAD6的突变 或Rev3基因将被引入以揭示活跃的突变 系统促成了“非程序化”的基因变化。

Spontaneous mutations contribute significantly to human genetic disease and form the background against which the genetic toxicity of environmental agents must be judged. In spite of these central concerns, the cellular processes that influence the spontaneous mutation rate are poorly understood. This project addresses key issues regarding the biochemical mechanisms of spontaneous mutagenesis eukaryotes: Is spontaneous mutation affected by processing of a gene by transcription and replication (Aim 1)? Is spontaneous mutation driven by metabolically generated DNA damage such as oxygen radicals (Aims 2-4)? Do repairable spontaneous DNA damages escape correction and cause mutations in normal cells (Aim 3)? Is their probability of generating mutations more a question of kinetics (repair vs. replication) or the rate at which damage is generated (Aim 4)? Do mutations due to endogenous DNA damage result from active processing pathways that handle environmental mutagens (Aim 5)? To achieve these ends, we will exploit our ability to modulate the DNA repair capacity of the yeast Saccharomyces cerevisiae by inactivating the APN1 gene we cloned previously, which encodes the main endonuclease for abasic sites in this organism. Strains lacking this activity have a substantially elevated rate of spontaneous mutation, which is generated in part by abasic sites produced by a DNA glycosylase (the MAG gene product) acting on endogenous DNA damages. We will construct test strains bearing a mutation targets of human origin, in common with the other projects. Rapid determination of mutation spectra using a newly-developed approach will allow us to tease out specific events that limit normal genetic stability. These targets will be inserted with different orientations respective to replication and transcription (leading vs. lagging strand, and transcribed vs. nontranscribed strand). The role of endogenous damage will be assessed by deleting the APN1 or MAG genes, and by culturing the cells under different conditions (aerobic vs. anaerobic, etc.). Yeast strains will also be constructed with increased levels of Apn1 protein or the human abasic endonuclease, Ape, which will reveal whether repair of such damages is limiting in normal cells. Finally, mutations in the RAD6 or REV3 genes will be introduced to reveal whether active mutational systems contribute to "unprogrammed" genetic change.