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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557210/
• 关键词: DnaA protein; Staphylococcus aureus; ATP-binding; basic phospholipid; acidic phospholipid; DNA replication; DNA polymerase III; silkworm; 大腸菌; DnaA蛋白質; 阻害剤; 抗菌物質; 抗菌剤; ATP; 蛋白質の精製

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.

金黄色葡萄球菌会导致人类机会性疾病。在这项研究中，我们检查了复制启动子金黄色葡萄球菌 DnaA 蛋白的生化性质。先前对大肠杆菌 DnaA 蛋白的研究表明，DnaA 蛋白的 ATP 结合形式具有活性，而 ADP 结合形式则无活性。纯化的金黄色葡萄球菌 DnaA 蛋白也表现出对 ATP 和 ADP 的高亲和力。我们发现磷脂酰甘油（一种酸性磷脂）会刺激 ADP-DnaA 复合物中 ADP 的释放，从而激活 DnaA 蛋白。赖氨酰磷脂酰甘油是一种碱性磷脂，可抑制磷脂酰甘油的作用。因此，磷脂酰甘油可能负向调节 DnaA 蛋白的重新激活。我们在这里提出了一种通过增加细胞质膜中碱性磷脂含量来调节DNA复制起始的新模型。我们分离了金黄色葡萄球菌的突变体，其DNA复制对温度敏感。通过互补分析，我们鉴定了细菌中 DNA 复制所必需的 polC、dnaE 和 dnaC 基因。我们还利用蚕建立了传染病动物模型。该系统将为对抗传染病的药物提供有用的筛选系统。

项目成果

Kubota T. et al.: "DnaA protein Lys-415 is close to the ATP-binding site : ATP-pyridoxal affinity labeling"Biochem Biophys Res Commun.. 288・5. 1141-1148 (2001)

Kubota T.等人：“DnaA蛋白Lys-415接近ATP结合位点：ATP-吡哆醛亲和标记”Biochem Biophys Res Commun. 288・5（2001）。

Kondo T.et al.: "Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNA supercoiling"Biochem J.. 348・2. 375-379 (2000)

Kondo T.等人：“通过过度DNA超螺旋抑制dnaA46突变体的温度敏感性”Biochem J.. 348・2 (2000)。

Kondo T. et al.: "Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNA supercoiling"Biochem J.. 348. 375-379 (2000)

Kondo T.等人：“通过过度DNA超螺旋抑制dnaA46突变体的温度敏感性”Biochem J.. 348. 375-379 (2000)

Nishida S. et al.: "A nucleotide switch in the E.coli DnaA protein initiates chromosomal replication : Evidence from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in vivo"J Biol Chem. (in press). (2002)

Nishida S. 等人：“大肠杆菌 DnaA 蛋白中的核苷酸开关启动染色体复制：来自体外和体内调节 ATP 水解缺陷的突变 DnaA 蛋白的证据”J Biol Chem。

Kuroda M. et al.: "Whole genome sequencing of meticillin-resistant Staphylococcus aureus"Lancet. 357. 1225-1240 (2001)

Kuroda M.等：“耐甲氧西林金黄色葡萄球菌的全基因组测序”《柳叶刀》。