NOVEL ENZYME ACTING ON OXIDATIVE DNA DAMAGE

作用于 DNA 氧化损伤的新型酶

• 批准号: 2700438
• 负责人: WILLIAM A FRANKLIN
• 金额: $ 23.07万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2700438
• 关键词: DNA binding protein; DNA damage; DNA repair; Escherichia coli; Schizosaccharomyces pombe; affinity chromatography; bacterial genetics; bacterial proteins; cofactor; complementary DNA; enzyme activity; exonuclease; genetic library; high performance liquid chromatography; intermolecular interaction; mutant; nucleic acid probes; oligonucleotides; plasmids; polymerase chain reaction; protein sequence

This is a revised application which is focused on the analysis of the mechanism of dRpase activity in bacteria, and the identification of corresponding activities in yeast. The specific aims are: (1) A novel plasmid assay construct will be used to analyze the role of the several known dRpase activities in bacterial DNA base excision repair. The plasmid assay construct will also be used to determine if the repair process involves replacement of one or several nucleotides. (2) Interactions between several proteins that function as part of the DNA base excision repair pathway will be investigated by use of the yeast two- hybrid system, and where appropriate by oligo-histidine affinity chromatography. Interactions will also be studied with purified repair enzymes and the plasmic assay system of SA1, in vitro, by analysis of repair rates as a function of added protein co-factors. (3) Characterization and purification of a eukaryotic dRpase activity in the yeast S. pombe. The yeast enzyme will be purified and subsequently subjected to peptide sequencing so as to yield oligonucleotide probes which can be used to screen a yeast cDNA library for the corresponding mRNA. It is proposed that this would allow for the sequencing, cloning, and possible source to identify a homologous human allele.

这是一个修订后的应用，重点是分析 DRPase活性在细菌中的机制和鉴定 酵母中的相应活动。 具体目的是：（1）小说 质粒测定构建体将用于分析几种的作用 在细菌DNA碱基切除修复中已知的DRPase活性。 这 质粒测定构建体还将用于确定是否维修 过程涉及替换一个或几个核苷酸。 （2） 几种起作用DNA一部分的蛋白质之间的相互作用 碱切除修复途径将通过使用两种酵母 混合动力系统，以及寡素 - 基斯丁亲和力的适当情况 色谱法。 还将通过纯化的维修研究相互作用 通过分析 维修速率与添加蛋白辅助因子的函数。 （3） 真核DRPase活性的表征和纯化 酵母S. Pombe。 酵母酶将被纯化，然后随后 进行肽测序以产生寡核苷酸探针 可用于筛选相应的酵母cDNA库 mRNA。 建议这将允许测序，克隆， 并可能识别同源人类等位基因的可能来源。