DNA TOPOISOMERASES AND OTHER ENZYMES AFFECTING CHROMATIN

DNA 拓扑异构酶和其他影响染色质的酶

• 批准号: 3275509
• 负责人: Rolf Sternglanz
• 金额: $ 16.99万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3275509
• 关键词: DNA directed RNA polymerase; DNA repair; DNA topoisomerases; Escherichia coli; acetylation; bacterial virus; chromatin; enzyme mechanism; fungal genetics; gene expression; genetic manipulation; genetic mapping; genetic recombination; genetic transcription; histones; messenger RNA; microorganism metabolism; molecular cloning; mutagen testing; nucleic acid hybridization; temperature sensitive mutant; transferase

A major aim is to continue studies on the in vivo roles of eukaryotic DNA topoisomerases using yeast topoisomerase mutants and genes isolated previously. Five separate projects are proposed: 1) to determine the role of topoisomerases in ribosomal RNA synthesis, 2) to learn why overproduction of DNA topoisomerase II is deleterious to yeast, 3) to clone human topoisomerase I and II genes by direct selection in yeast. A human cDNA library will be placed in a yeast expression vector plasmid. The DNA will be used to transform a yeast mutant deficient in topoisomerase I and II; rare transformants which have gained topoisomerase I or II activity will be selected by their more rapid growth. DNA topoisomerases are known to be the targets of a wide variety of anti-cancer drugs. Isolation of their genes will allow overproduction of the enzymes, which will be useful for testing new derivatives of the known drugs, 4) to determine if the in vivo target of the drug novobiocin in yeast is topoisomerase II or some other enzyme, and 5) to determine if RNA polymerase II, the enzyme responsible for mRNA synthesis, has topoisomerase activity inherent in it, distinct from the known topoisomerases I and II. Another major aim is to find a yeast mutant deficient in histone acetyl transferase and to use the mutant to determine the role of histone acetylation in chromatin structure and function. The approach will be the same as was used successfully to identify yeast topoisomerase mutants, namely, to screen a collection of heavily mutagenized temperature-sensitive mutants by direct enzymatic assay. Once a mutant is identified, its phenotypes will be studied to learn about the in vivo role of the enzyme.

主要目标是继续研究真核 DNA 的体内作用 使用酵母拓扑异构酶突变体和分离的基因的拓扑异构酶 之前。 提出了五个单独的项目：1）确定角色 拓扑异构酶在核糖体 RNA 合成中的作用，2) 了解原因 DNA 拓扑异构酶 II 的过量产生对酵母有害，3) 克隆 通过在酵母中直接选择人类拓扑异构酶 I 和 II 基因。 一个人 将cDNA文库置于酵母表达载体质粒中。 脱氧核糖核酸 将用于转化拓扑异构酶 I 缺陷的酵母突变体 二、获得拓扑异构酶 I 或 II 活性的罕见转化体 将因其更快的成长而被选择。 DNA拓扑异构酶是已知的 成为多种抗癌药物的靶标。 隔离 他们的基因将允许酶的过量生产，这将是有用的 用于测试已知药物的新衍生物，4) 确定是否 酵母中药物新生霉素的体内靶标是拓扑异构酶 II 或某些酶 其他酶，以及 5) 确定 RNA 聚合酶 II 是否为该酶 负责 mRNA 合成，具有固有的拓扑异构酶活性， 与已知的拓扑异构酶 I 和 II 不同。 另一个主要目标是找到缺乏组蛋白乙酰基的酵母突变体 转移酶并使用突变体来确定组蛋白的作用 染色质结构和功能中的乙酰化。 该方法将是 与成功用于鉴定酵母拓扑异构酶突变体的方法相同， 即筛选一系列经过严重诱变的温度敏感的 通过直接酶法测定突变体。 一旦突变体被识别出来， 将研究表型以了解酶的体内作用。