Interaction between the replicational initiator DnaA and the DNA polymerase components

复制起始子 DnaA 和 DNA 聚合酶组分之间的相互作用

• 批准号: 10680616
• 负责人: KATAYAMA Tsutomu
• 金额: $ 2.18万
• 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: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680616/
• 关键词: chromosome; DNA replication; ATP; DnaA; DNA polymerase; Sliding clamp; Replicational initiation; Cell cycle; スライディング・クランプ; 染色体複製; otiC; 大腸菌

In E. coli, DnaA protein forms a complex with the chromosomal replication origin (oriC). In this complex, DnaA opens the duplex DNA. This initiation activity of DnaA protein depends on ATP binding. On the generated single stranded region, DNA polymerase III, the replicase of chromosome, is loaded.We recently revealed that the hydrolysis of ATP bound to DnaA is accelerated by interaction with the sliding clamp of DNA polymerase III and a partially purified IdaB protein fraction. The sliding clamp is a ring-like configuration that is comprised from a dimer of the β subunit of the replicase and tethers DNA strand to the replicase. By this reaction, the ADP form of DnaA, an inactive form for initiation, is rapidly generated. We named this system RIDA for Regulatory Inactivation of DnaA, and suggested that RIDA is a main system to prevent extra initiations for maintaining the frequency of initiation only once per chromosome per cell cycle. Indeed, certain DnaA mutant proteins that are insensitive to a RIDA causes overinitiation of chromosomal replication. Moreover, we had in vivo evidence that RIDA depends on genes necessary for DNA replication. Content of the ATP form of DnaA in randomly dividing cells is 15 - 30% of total ATP/ADP-bound DnaA molecules, and when genes required for DNA replication were inactivated, the ATP-DnaA level increased up to 80%. These results suggest that the ATP-DnaA level is restrained during the cell cycle depending on DNA replication, which may be a key event to control the initiation cycle of chromosomal replication.

在大肠杆菌中，Dna A蛋白与染色体复制起点(ORIC)形成复合体。在这个复合体中，DNAA打开双链DNA。DNAA蛋白的这种启动活性依赖于与ATP的结合。在产生的单链区，加载了染色体的复制酶DNA聚合酶III。我们最近发现，通过与DNA聚合酶III的滑动夹和部分纯化的IdAB蛋白片段的相互作用，与DNAA结合的ATP的水解被加速。滑动夹是由复制酶的DNA亚单位的二聚体组成的环状构型，并将β链系在复制酶上。通过这个反应，DNAA的ADP形式迅速产生，DNAA是一种不活跃的引发形式。我们将这个系统命名为RIDA，用于DNAA的调节失活，并建议RIDA是防止额外启动的主要系统，以保持每个染色体在每个细胞周期中只有一次启动频率。事实上，某些对RIDA不敏感的DNAA突变蛋白会导致染色体复制的过度启动。此外，我们有体内证据表明，RIDA依赖于DNA复制所必需的基因。在随机分裂的细胞中，ATP型DNAA的含量占与ATP/ADP结合的DNAA分子总数的15%-30%，当DNA复制所需的基因失活时，ATP-DNAA水平上升到80%。这些结果表明，在细胞周期中，ATP-DNAA水平受到依赖于DNA复制的抑制，这可能是控制染色体复制起始周期的关键事件。

项目成果

Katayama, T.: "Novel function of DNA polymerase : Negative regulation for initiation of the chromosomal replication"Protein-Nucleic acid -Enzyme. 44(7). 845-853 (1999)

Katayama, T.：“DNA 聚合酶的新功能：染色体复制起始的负调控”蛋白质核酸酶。

Katayama, T., Sekimizv, K: "Inactivation of Escherichia coli DnaA protein by DNA polymerase III, and negative regulations for initiation of chromosomal replication."Biochimie. 81. 835-840 (1999)

Katayama, T., Sekimizv, K：“DNA 聚合酶 III 灭活大肠杆菌 DnaA 蛋白，以及染色体复制起始的负调控。”Biochimie。

Kurokawa, K., Nishida, S., Emoto, A., Sekimizu, K., and Katayama, T.: "Replication cycle-coordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherichia coli"EMBO J.. 18(23). 6642-6652 (1999)

Kurokawa, K.、Nishida, S.、Emoto, A.、Sekimizu, K. 和 Katayama, T.：“大肠杆菌中 DnaA 蛋白的腺嘌呤核苷酸结合形式的复制周期协调变化”EMBO J..

Takata,M,Gvo,L.,Katayama,T.,Hase,,M.,Seyama,Y.,Miki,T.,Sekimizu,K.: "Mutant DnaA proteins defective in opening of oriC, the origin of chromosomal DNA replication in Escherichia coli"Mal.Microbol. 35・.2. 454-462 (2000)

Takata, M.、Gvo, L.、Katayama, T.、Hase, M.、Seyama, Y.、Miki, T.、Sekimizu, K.：“在 oriC（染色体 DNA 的起源）的开放中存在缺陷的突变 DnaA 蛋白在大肠杆菌中的复制“Mal.Microbol.35・.2.454-462(2000)

片山 勉: "DNAポリメラーゼの新機能:染色体複製開始の負の制御" 蛋白質核酸酵素. 印刷中.

Tsutomu Katayama：“DNA聚合酶的新功能：染色体复制起始的负调节”蛋白质核酸酶正在出版。