BIOCHEMICAL CHARACTERIZATION OF YEAST DNA HELICASES

酵母 DNA 解旋酶的生化特征

• 批准号: 2187994
• 负责人: STEVEN W MATSON
• 金额: $ 13.04万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187994
• 关键词: DNA; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; chemical kinetics; computer assisted sequence analysis; enzyme mechanism; enzyme structure; enzyme substrate; fungal genetics; helicase; molecular cloning; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; protein purification; protein sequence; western blottings

DESCRIPTION (Adapted from the applicant's abstract): The double stranded structure of DNA mandates the existence of a mechanism for unwinding the helix to expose single-stranded DNA (ssDNA) for use as a template or reaction intermediate in DNA replication, repair, recombination and perhaps transcription. The DNA helicases are enzymes which provide such a mechanism. These enzymes translocate through duplex DNA disrupting the hydrogen bonds that hold the two strands together in a reaction utilizing energy provided by NTP hydrolysis. More than ten DNA helicases have been described in the prokaryote E. coli; the role of each in DNA metabolism is currently being elucidated using a combination of biochemical and genetic studies. To date comparable detailed studies of DNA helicases in eukaryotic organisms have lagged behind. Recently, the principal investigator initiated a project aimed toward identifying and characterizing DNA helicases from the budding yeast Saccharomyces cerevisiae. Yeast has been chosen as an example of a eukaryotic organism that is amenable to both biochemical and genetic studies. Four previously undescribed DNA helicases have been identified by chemical criteria; three have been purified to apparent homogeneity. The long range goal of this project is to characterize, biochemically and genetically, the four new yeast DNA helicases that have been isolated. Each of these enzymes will be purified to homogeneity and characterized with respect to DNA/RNA substrate requirements, reaction mechanism (processive versus distributive), and interactions with other proteins. Enough protein will be purified to obtain sufficient amino acid sequence data to allow cloning of the gene encoding each enzyme. This will permit a genetic analysis to begin to elucidate the precise biochemical function of each of these helicases in the cell.

描述（改编自申请人的摘要）： DNA的结构要求存在一种机制， 螺旋以暴露单链DNA（ssDNA）用作模板，或 DNA复制、修复、重组的反应中间体， 转录。 DNA解旋酶是提供这种解旋的酶。 机制 这些酶通过双链体DNA易位， 在反应中将两条链结合在一起的氢键， NTP水解提供的能量。 已经有十多个DNA解旋酶 在原核生物E.大肠杆菌中;每一种在DNA代谢中的作用是 目前正在使用生物化学和遗传学的结合来阐明 问题研究 到目前为止，对DNA解旋酶的比较详细的研究， 真核生物已经落后了。 最近，校长 研究人员启动了一个项目，旨在确定和 芽殖酵母属DNA解旋酶的特性 啤酒。 酵母被选为真核生物的一个例子 可以进行生物化学和遗传学研究。 四个先前 未描述的DNA解旋酶已确定的化学标准;三个 已经被提纯到表面上的同质性。 长期目标是 该项目是表征，生物化学和遗传，四个新的 已分离的酵母DNA解旋酶。 这些酶中的每一种都会 纯化至均一并对DNA/RNA底物进行表征 需求、反应机制（过程性与分配性），以及 与其他蛋白质的相互作用。 足够的蛋白质将被纯化， 获得足够的氨基酸序列数据以允许基因的克隆 编码每一种酶。 这将允许遗传分析开始， 阐明这些解旋酶中每一种的精确生物化学功能， 牢房