Molecular design of inhibitors for DNA replication in Escherichia coli

细胞内DNA复制的启动受到多种因素的积极或消极调节。我们试图通过生物化学和遗传学的方法来确定这些调节因子。在大肠杆菌的粗提物中，我们发现了一种活性（RIDA，调控抑制DnaA蛋白），它可以刺激与DnaA蛋白结合的ATP水解，而DnaA蛋白是大肠杆菌DNA复制的起始物。该因子的纯化表明，DNA聚合酶III全酶的β亚基负责该活性。在编码β亚基的dnaN基因突变细胞中，对DNA复制蛋白无活性的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ATP与dna蛋白结合的水解。这是首次报道DNA聚合酶的一个亚基负调控DNA复制的起始，该亚基负责DNA合成的催化反应。

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.