Katayama T, Akimitsu N, Mizushima T, Miki T, Sekimizu K
Department of Microbiology, Kyushu University Faculty of Pharmaceutical Sciences, Fukuoka, Japan.
Mol Microbiol. 1997 Aug;25(4):661-70. doi: 10.1046/j.1365-2958.1997.5001872.x.
The activity of DnaA protein, the initiator of chromosome replication in Escherichia coli, is regulated by adenine nucleotide binding; the ATP-bound form, not the ADP-bound form, is active. DnaAcos is a mutant protein that is insensitive to negative regulation by ADP. Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30 degrees C, a restrictive temperature for growth. To determine the control factors that act independently of adenine nucleotide binding of DnaA, we analysed suppressors from the dnaAcos mutant isolated by Tn5 insertion mutagenesis. Three of the suppressors carried Tn5 in the aroK or aroB gene, the first two cistrons in the dam operon. Complementation tests revealed that the dam gene is responsible for the suppression. Over-replication of the chromosome was inhibited in the dnaAcos aroK::Tn5 double mutant, and initiation of chromosome replication in the dnaA+ aroK::Tn5 mutant was partially inhibited. The aroK(or B)::Tn5 cells contained DnaA molecules at a level similar to that in the parental aroBK+ strain. Moreover, dnaAcos suppression depended on the function of the seqA gene. Thus, Dam activity positively regulates initiation of chromosome replication in vivo. SeqA function seems to be distinguished from the control of DnaA protein by adenine nucleotide binding.
DnaA蛋白是大肠杆菌染色体复制的起始因子，其活性受腺嘌呤核苷酸结合调控；与ATP结合的形式有活性，与ADP结合的形式无活性。DnaAcos是一种对ADP的负调控不敏感的突变蛋白。在30℃（生长的限制温度）下，dnaAcos突变体中染色体复制起始过度。为了确定独立于DnaA腺嘌呤核苷酸结合起作用的控制因子，我们分析了通过Tn5插入诱变从dnaAcos突变体中分离出的抑制子。其中三个抑制子在aroK或aroB基因中携带Tn5，aroK或aroB基因是dam操纵子的前两个顺反子。互补试验表明dam基因负责这种抑制作用。在dnaAcos aroK::Tn5双突变体中，染色体的过度复制受到抑制，在dnaA+ aroK::Tn5突变体中，染色体复制起始受到部分抑制。aroK（或B）::Tn5细胞中DnaA分子的水平与亲本aroBK+菌株中的水平相似。此外，dnaAcos抑制作用依赖于seqA基因的功能。因此，Dam活性在体内对染色体复制起始起正调控作用。SeqA的功能似乎与通过腺嘌呤核苷酸结合对DnaA蛋白的调控不同。
