Katayama T, Kornberg A
Department of Biochemistry, Stanford University School of Medicine, California 94305.
J Biol Chem. 1994 Apr 29;269(17):12698-703.
DnaA protein initiates genomic replication in Escherichia coli. A cold-sensitive dnaAcos mutant caused excessive initiation at a restrictive temperature without an increase in the level of DnaA protein. The chromosomal origin (oriC) was essential for the lethality caused by the dnaAcos product. Increased initiation activity was neutralized by multiple copies of oriC on a plasmid (pBR322). OriC plasmids were replicated efficiently in vitro in a crude extract prepared from a dnaAcos mutant, with a specific activity for the DnaAcos protein 8-fold greater than that for the DnaA+ protein in a wild-type extract. OriC-dependent replication in the dnaAcos extract was inhibited by rifampicin and by gyrase inhibitors as was replication in the dnaA+ extract. As a control, replication of single-stranded phage phi X174 DNA, which did not require DnaA protein, was similar in extracts prepared from dnaA+ and dnaAcos cells. Thus, initiation at oriC by DnaAcos protein appears to be highly activated both in vivo and in vitro.
DnaA蛋白启动大肠杆菌中的基因组复制。一个冷敏感的dnaAcos突变体在限制温度下导致过度起始，而DnaA蛋白水平并未增加。染色体复制起点（oriC）对于dnaAcos产物引起的致死性至关重要。质粒（pBR322）上多个拷贝的oriC可中和增加的起始活性。oriC质粒在由dnaAcos突变体制备的粗提取物中能在体外高效复制，其中DnaAcos蛋白的比活性比野生型提取物中DnaA +蛋白的比活性高8倍。如在DnaA +提取物中一样，rifampicin和gyrase抑制剂可抑制dnaAcos提取物中依赖oriC的复制。作为对照，单链噬菌体phi X174 DNA的复制不需要DnaA蛋白，在由DnaA +和dnaAcos细胞制备的提取物中相似。因此，DnaAcos蛋白在oriC处的起始似乎在体内和体外均被高度激活。
