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: 3275510
• 负责人: Rolf Sternglanz
• 金额: $ 17.39万
• 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/3275510
• 关键词: DNA directed RNA polymerase; DNA repair; DNA replication; DNA topoisomerases; Escherichia coli; RNA biosynthesis; 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.

一个主要目的是继续研究真核DNA在体内的作用 使用酵母拓扑异构酶突变体的拓扑异构酶和分离的基因 以前。 提出了五个单独的项目：1）确定作用 核糖体RNA合成中的拓扑异构酶，2）了解为什么 过量产生DNA拓扑异构酶II对酵母有害，3）克隆 人拓扑异构酶I和II基因。 人类 将cDNA文库置于酵母表达载体质粒中。 的DNA 将用于转化拓扑异构酶I缺陷的酵母突变体， II;获得拓扑异构酶I或II活性的罕见转化体 会被他们更快的成长所选中。 已知DNA拓扑异构酶 成为各种抗癌药物的靶点。 分离 他们的基因将允许酶的过量生产，这将是有用的 为了测试已知药物的新衍生物，4）确定是否在 新生霉素在酵母中的体内靶点是拓扑异构酶II或一些 5）确定RNA聚合酶II，所述酶 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。 另一个主要目的是找到一个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。 方法将是 与成功用于鉴定酵母拓扑异构酶突变体相同， 也就是说，筛选一批高度诱变的温度敏感的 通过直接酶法测定突变体。 一旦发现突变体， 将研究表型以了解酶的体内作用。