REGULATORY MACHINERY FOR THE E. COLI CHROMOSOMAL REPLICATION CYCLE

大肠杆菌染色体复制周期的调节机制

• 批准号: 12680677
• 负责人: KATAYAMA Tsutomu
• 金额: $ 2.5万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 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/grant/KAKENHI-PROJECT-12680677/
• 关键词: DNA Replication; Cell Cycle; DNA Polymerase; DnaA Protein; ATP; AAA+Family; Chromosome; E. Coli; 染色体複製開始制御; ゲノム

In Escherichia coli, ATP-bound DnaA protein forms initiation complex with the replicational origin for chromosomal replication. I found that hydrolysis of DnaA-bound ATP is promoted by a specific DNA-protein complex called the sliding clamp which is formed upon loading of the DNA polymerase III (the chromosomal replicase) onto DNA. The resultant ADP-DnaA is inactive for initiation, and thus overinitiation is repressed (Cell, 1998; EMBO J., 1999). In this research program, I identified a novel clue for this system termed RIDA (regulatory inactivation of DnaA). This clue termed IdaB/Hda protein is required for RIDA in vitro and in vivo. Using purified IdaB/Hda protein, the sliding clamp, and DnaA protein, I succeeded construction of in vitro reconstituted RIDA system (EMBO J., 2001). Using this and other experimental systems, I revealed functional structure and structure-function relationship of the sliding clamp and DnaA protein in RIDA reaction. Furthermore, I search for factors re-activating ADP-DnaA protein, and found a specific DNA sequence including DnaA-binding motif as an activator. As such, this research program was carried out with important progress in analyses of DnaA regulation and the replication cycle control.

在大肠杆菌中，ATP 结合的 DnaA 蛋白与染色体复制的复制起点形成起始复合物。我发现 DnaA 结合的 ATP 的水解是由一种称为滑动夹的特定 DNA-蛋白质复合物促进的，该复合物是在 DNA 聚合酶 III（染色体复制酶）加载到 DNA 上时形成的。所得的 ADP-DnaA 对于启动没有活性，因此过度启动受到抑制（Cell，1998；EMBO J.，1999）。在这个研究项目中，我发现了这个系统的一个新线索，称为 RIDA（DnaA 的调节失活）。这种称为 IdaB/Hda 蛋白的线索是体外和体内 RIDA 所必需的。使用纯化的IdaB/Hda蛋白、滑夹和DnaA蛋白，我成功构建了体外重组RIDA系统（EMBO J.，2001）。使用这个和其他实验系统，我揭示了 RIDA 反应中滑动夹和 DnaA 蛋白的功能结构和结构功能关系。此外，我寻找重新激活ADP-DnaA蛋白的因子，并发现了包含DnaA结合基序的特定DNA序列作为激活剂。因此，该研究计划在 DnaA 调控和复制周期控制分析方面取得了重要进展。

项目成果

Nishida, S., Fujimitsu, K., Sekimizu, K., Ohmura, T., Ueda, T., Katayama, T.: "A nucleotide switch in E. coli DnaA protein initiates the chromosomal replication : Evidence from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in v

Nishida, S.、Fujimitsu, K.、Sekimizu, K.、Ohmura, T.、Ueda, T.、Katayama, T.：“大肠杆菌 DnaA 蛋白中的核苷酸开关启动染色体复制：来自突变体 DnaA 的证据

Nishida, S., Fujimitsu, K., Sekimizu, K., Ohmura, T., Ueda, T., Katayama, T.: "A nucleotide switch in E. coli DnaA protein initiates the chromosomal replication : Evidence from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in v

Nishida, S.、Fujimitsu, K.、Sekimizu, K.、Ohmura, T.、Ueda, T.、Katayama, T.：“大肠杆菌 DnaA 蛋白中的核苷酸开关启动染色体复制：来自突变体 DnaA 的证据

Kawakami, H., Iwura, T., Takata, M., Sekimizu, K., Hiraga, S., Katayama, T.: "Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the reolicase and DnaA protein"Mol. Genet. Genomics.

Kawakami, H.、Iwura, T.、Takata, M.、Sekimizu, K.、Hiraga, S.、Katayama, T.：“通过 reolicase 和 DnaA 滑动夹中的遗传改变来阻止细胞分裂和类核分配

Kato, J., Katayama, T.: "Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli"EMBO Journal.

Kato, J.，Katayama, T.：“Hda，一种新型 DnaA 相关蛋白，调节大肠杆菌的复制周期”EMBO 杂志。

Kato, J. and Katayama, T.: "Hda, a novel DnaArelated protein, regulates the replication cycle in Escherichia coli"EMBO J.. 20(15). 4253-4262 (2001)

Kato, J. 和 Katayama, T.：“Hda 是一种新型 DnaA 相关蛋白，调节大肠杆菌的复制周期”EMBO J.. 20(15)。