Hyperactive initiation of chromosomal replication in vivo and in vitro by a mutant initiator protein, DnaAcos, of Escherichia coli

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 φX174 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.