Takata M, Guo L, Katayama T, Hase M, Seyama Y, Miki T, Sekimizu K
Department of Molecular Microbiology, Kyushu University Graduate School of Pharmaceutical Sciences, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
Mol Microbiol. 2000 Jan;35(2):454-62. doi: 10.1046/j.1365-2958.2000.01722.x.
We characterized three mutant DnaA proteins with an amino acid substitution of R334H, R342H and E361G that renders chromosomal replication cold (20 degrees C) sensitive. Each mutant DnaA protein was highly purified from overproducers, and replication activities were assayed in in vitro oriC replication systems. At 30 degrees C, all three mutant proteins exhibited specific activity similar to that seen with the wild-type protein, whereas at 20 degrees C, there was much less activity in a replication system using a crude replicative extract. Regarding the affinity for ATP, the dissociation rate of bound ATP and binding to oriC DNA, the three mutant DnaA proteins showed a capacity indistinguishable from that of the wild-type DnaA protein. Activity for oriC DNA unwinding of the two mutant DnaA proteins, R334H and R342H, was more sensitive to low temperature than that of the wild-type DnaA protein. We propose that R334H and R342H have a defect in their potential to unwind oriC DNA at low temperatures, the result being the cold-sensitive phenotype in oriC DNA replication. The two amino acid residues of DnaA protein, located in a motif homologous to that of NtrC protein, may play a role in the formation of the open complex. The E361 residue may be related to interaction with another protein present in a crude cell extract.
我们对三种突变型DnaA蛋白进行了表征，它们分别发生了R334H、R342H和E361G的氨基酸替换，这些替换使得染色体复制对低温（20摄氏度）敏感。每种突变型DnaA蛋白都从过量表达菌株中高度纯化出来，并在体外oriC复制系统中测定复制活性。在30摄氏度时，所有三种突变蛋白的比活性与野生型蛋白相似，而在20摄氏度时，使用粗制复制提取物的复制系统中的活性则低得多。关于对ATP的亲和力、结合的ATP的解离速率以及与oriC DNA的结合，这三种突变型DnaA蛋白表现出与野生型DnaA蛋白无法区分的能力。两种突变型DnaA蛋白R334H和R342H对oriC DNA解旋的活性比野生型DnaA蛋白对低温更敏感。我们提出，R334H和R342H在低温下解旋oriC DNA的潜力存在缺陷，结果是oriC DNA复制出现冷敏感表型。DnaA蛋白的两个氨基酸残基位于与NtrC蛋白同源的基序中，可能在开放复合物的形成中起作用。E361残基可能与与粗制细胞提取物中存在的另一种蛋白质的相互作用有关。
