Interaction between the replicational initiator DnaA and the DNA polymerase components

在大肠杆菌中，dna蛋白与染色体复制原点（oriC）形成复合体。在这个复合体中，DNA打开双链DNA。dna蛋白的起始活性取决于ATP的结合。在生成的单链区域，装载了DNA聚合酶III，即染色体复制酶。我们最近发现，通过与DNA聚合酶III的滑动钳和部分纯化的IdaB蛋白片段相互作用，ATP与DNA结合的水解可以加速。滑动钳是一种环状结构，由复制酶的β亚基二聚体组成，将DNA链系在复制酶上。通过这个反应，dna的ADP形式，一种起始的无活性形式，被迅速生成。我们将该系统命名为RIDA，即dna的调节失活，并认为RIDA是防止额外起始的主要系统，以维持每个细胞周期每条染色体仅起始一次的频率。事实上，某些对RIDA不敏感的dna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。随机分裂细胞中ATP形式的DNA含量占总ATP/ adp结合DNA分子的15 - 30%，当DNA复制所需基因失活时，ATP-DnaA含量可增加80%。这些结果表明，atp - DNA水平在细胞周期中受到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。

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.