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Development of antibiotics by monitoring the inhibition of the ATP-binding to DnaA, the initiator protein of chromosomal DNA replication in bacteria

通过监测 ATP 与 DnaA 结合的抑制来开发抗生素，DnaA 是细菌中染色体 DNA 复制的起始蛋白

基本信息

批准号：
12557210
负责人：
SEKIMIZU Kazuhisa
金额：
$ 7.68万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Staphylococcus aureus causes opportunistic diseases for humans. In this study, we examined biochemical nature of Staphylococcus aureus DnaA protein, the replication initiator. Previous studies with Escherichia coli DnaA protein demonstrated that the ATP-binding form of DnaA protein is active, whereas the ADP-binding form is inactive. Purified Staphylococcus aureus DnaA proteins also showed high affinity for ATP and ADP. We showed that phosphatidylglycerol, an acidic phospholipids, stimulated the release of ADP from the ADP-DnaA complex, resulting in the activation of DnaA protein. Lysylphosphatidylglycerol, a basic phospholipid, was shown to inhibit the action of phosphatidylglycerol. Thus, phosphatidylglycerol may negatively regulate re-activation of DnaA protein. We propose here a new model of the regulation of initiation of DNA replication by increase in the content of basic phospholipids in cytoplasmic membranes.We isolated mutants of Staphylococcus aureus whose DNA replication were temperature-sensitive. By complementation analysis, we identified polC, dnaE, and dnaC genes which are essential for DNA replication in the bacteria. We also established an animal model of infectious diseases by using silkworms. The system will provide a useful screening system for medicines against infectious diseases.

金黄色葡萄球菌引起人类机会性疾病。在这项研究中，我们检测了金黄色葡萄球菌dna蛋白的生化性质，dna蛋白是金黄色葡萄球菌复制的起始物。先前对大肠杆菌dna蛋白的研究表明，dna蛋白的atp结合形式是有活性的，而adp结合形式是无活性的。纯化后的金黄色葡萄球菌dna蛋白对ATP和ADP也表现出高亲和力。我们发现磷脂酰甘油，一种酸性磷脂，刺激ADP-DnaA复合物释放ADP，导致DnaA蛋白活化。赖氨酸磷脂酰甘油是一种碱性磷脂，具有抑制磷脂酰甘油作用的作用。因此，磷脂酰甘油可能负向调节dna蛋白的再激活。我们在这里提出了一个新的模型，通过增加细胞质膜中碱性磷脂的含量来调节DNA复制的起始。我们分离了DNA复制对温度敏感的金黄色葡萄球菌突变体。通过互补分析，我们鉴定出了polC、dnaE和dnaC基因，它们是细菌DNA复制所必需的基因。我们还利用家蚕建立了传染病动物模型。该系统将提供一个有用的传染病药物筛选系统。