T4 Phage dNTP Synthetase: A Multienzyme Complex for Deoxyribonucleotide Synthesis

T4 噬菌体 dNTP 合成酶：用于脱氧核糖核苷酸合成的多酶复合物

• 批准号: 8916366
• 负责人: Christopher Mathews
• 金额: $ 31.43万
• 依托单位: Oregon State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8916366&HistoricalAwards=false
• 关键词: T4; Phage; dNTP; Synthetase; Multienzyme

This research involves a multienzyme complex, formed in T4 phage- infected Escherichia coli, which carries out the synthesis of deoxyribonucleoside tripgisohates (dNTPs). This T4 dNTP synthesis complex will be investigated by both biochemical and genetic means, with the goals of learning how it functions and how it integrates dNTP synthesis with DNA replication. First, through analysis of complexes isolated from T4-infected cells and through partial reconstitution of the complex from purified proteins, we hope to define the protein composition, stoichiometry, and protein-protein interactions within the complex. Second, through assays for multi-step reaction pathways catalayzed by the purified complex, and through study of the allosteric properties of its constituent enzymes, we hope to learn how regulation of individual enzymes contributes toward control of the complex, and in turn helps to explain how dNTP synthesis is controlled in vitro. Third, through enzyme kinetic analysis, studies with bifunctional protein crosslinkers, and investigations of dNTP pool dynamics and DNA synthesis in vitro, we hope to learn whether the dNTP synthetase complex is physically or functionally linked to the DNA replication apparatus in vivo. Fourth, through reversion analysis at defined loci in the T4 rII cistrons, we will investigate mechanisms of mutagenesis induced by DNA precursor pool imbalances, with some of the data possibly generating insight into the nature of the microenvironment at DNA replication sites.

本研究涉及在T4噬菌体感染的大肠杆菌中形成的多酶复合物，该复合物进行脱氧核糖核苷三甘油三酯（dNTPs）的合成。该T4 dNTP合成复合物将通过生化和遗传手段进行研究，目的是了解其功能以及如何将dNTP合成与DNA复制相结合。首先，通过分析从t4感染细胞中分离的复合物，并通过纯化蛋白对复合物进行部分重构，我们希望确定复合物内的蛋白质组成、化学计量学和蛋白质-蛋白质相互作用。其次，通过对纯化络合物催化的多步反应途径的分析，并通过对其组成酶的变构性质的研究，我们希望了解单个酶的调控如何有助于对络合物的控制，进而有助于解释dNTP的合成是如何在体外控制的。第三，通过酶动力学分析、双功能蛋白交联剂的研究、dNTP池动力学和体外DNA合成的研究，我们希望了解dNTP合成酶复合物是否与体内DNA复制装置有物理或功能上的联系。第四，通过对T4 - rII顺子中特定位点的逆转分析，我们将研究由DNA前体池失衡引起的诱变机制，其中一些数据可能有助于了解DNA复制位点微环境的性质。