NOVEL ENZYME ACTING ON OXIDATIVE DNA DAMAGE

作用于 DNA 氧化损伤的新型酶

• 批准号: 2894832
• 负责人: WILLIAM A FRANKLIN
• 金额: $ 24万
• 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/2894832
• 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程序，重点是分析 细菌 dRpase 活性的机制及鉴定 酵母中的相应活性。 具体目标是：（1）小说 质粒测定结构将用于分析几个 已知 dRpase 在细菌 DNA 碱基切除修复中的活性。 这 质粒测定构建体也将用于确定修复是否 该过程涉及一个或多个核苷酸的替换。 (2) 作为 DNA 一部分的几种蛋白质之间的相互作用 碱基切除修复途径将通过使用酵母二- 混合系统，并在适当的情况下通过寡聚组氨酸亲和力 色谱法。 还将通过纯化修复来研究相互作用 酶和 SA1 的血浆测定系统，体外，通过分析 修复率作为添加的蛋白质辅因子的函数。 (3) 真核 dRpase 活性的表征和纯化 粟酒裂殖酵母。 酵母酶将被纯化，随后 进行肽测序以产生寡核苷酸探针 可用于筛选酵母 cDNA 文库中相应的 mRNA。 建议这将允许测序、克隆、 以及鉴定同源人类等位基因的可能来源。