REPAIR OF OXIDATIVE DNA DAMAGE

修复氧化性 DNA 损伤

• 批准号: 2565751
• 负责人: V A BOHR
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2565751
• 关键词: DNA damage; DNA repair; chromatography; guanosine; laboratory rat; mitochondrial DNA; oxidative stress; radiation genetics; tissue /cell culture

We have developed assays to detect oxidative lesions in specific genes and to quantitate their formation and repair. Oxidative DNA damage is generated by several different approaches including hydrogen peroxide, acridine orange, X-irradiation, irradiation with methylene blue. The main lesion which we examine is 8-OH guanosine which can be detected by use of the Fpg glycosylase. This enzyme creates strand breaks in DNA at sites of the lesions, and the single stranded DNA can then be resolved on alkaline gels. We find that 8-OH guanosine is rapidly repaired in active genes and in mitochondrial DNA. We have developed an in vitro assay for DNA nicking activity in mitochondrial extracts from rats. We have identified a protein from mitochondria which is able to incise an oligo containing a site specific 8-oxoguanine adduct. Unlike the bacterial enzyme which cleaves both 3' and 5' to the lesion, the mitochondrial enzyme incises exclusively on the 3' side of the lesion. We have finalized our chromatography steps and have begun the physical characterization of the enzyme.

我们已经开发了检测特定基因中氧化损伤的检测方法， 来量化它们的形成和修复。氧化性DNA损伤 由几种不同的方法产生，包括过氧化氢， 吖啶橙子、X射线照射、亚甲蓝照射。 主要 我们检查的病变是8-OH鸟苷，其可以通过使用 Fpg糖基化酶。 这种酶在DNA中产生链断裂， 病变的位点，然后可以将单链DNA解析到 碱性凝胶 我们发现，8-OH鸟苷是迅速修复，在积极的 基因和线粒体DNA。 我们已经开发了一种体外测定DNA切口活性的方法， 线粒体提取物。我们已经鉴定出一种蛋白质， 能够切割含有位点特异性寡核苷酸的线粒体 8-氧代鸟嘌呤加合物。不像细菌的酶， 在损伤的5'端，线粒体酶只在 病变的3'侧。我们已经完成了色谱步骤， 已经开始了酶的物理特性研究。