Molecular design of inhibitors for DNA replication in Escherichia coli

细胞中DNA复制的起始受多种因素的负或正调控。我们试图通过生物化学和遗传学的方法来鉴定这些调控因子。在大肠杆菌的粗提物中，我们鉴定了一种活性（RIDA，DnaA蛋白的调节抑制），其刺激与DnaA蛋白结合的ATP的水解，DnaA蛋白是大肠杆菌中DNA复制的起始物。该因子的纯化证明DNA聚合酶III全酶的β亚基负责该活性。在编码β亚基的dnaN基因的突变细胞中，对DNA复制无活性的DnaA蛋白的ADP结合形式被证明是积累的。结果证实β亚基刺激与DnaA蛋白结合的ATP的水解。这是第一份报告表明，DNA聚合酶的一个亚基，这是负责催化反应的DNA合成，负调控DNA复制的起始。

Initiation of DNA replication in cells is negatively or positively regulated by various factors. We tried to identify those regulating factors by using biochemical and genetic approaches. In crude extacts of E.coli, we identified an activity (RIDA, regulatory inhibition of DnaA protein) which stimulates hydrolysis of ATP bound to DnaA protein, the initiator of DNA replication in E coli. Purification of this factor demonstrated that the beta subunit of DNA polymerase III holoenzyme is responsible for this activity. In a mutant cells of the dnaN gene, which encodes the beta subunit, the ADP-binding form of DnaA protein, which is inactive for DNA replicatoin, was shown to be accumulated. The result confirms that the beta subunit stimulates hydrolysis of ATP bound to DnaA protein. This is the first report indicating that a subunit of DNA polymerase, which is responsible for catalytic reaction of DNA synthesis, negatively regulates initiation of DNA replication.