Inactivation Of DnaA Research Articles

The Escherichia coli datA site promotes proper regulation of cell division

In Escherichia coli inhibition of replication leads to a block of cell division. This checkpoint mechanism ensures that no cell divides without having two complete copies of the genome to pass on to the two daughter cells. The chromosomal datA site is a 1 kb region that contains binding sites for the DnaA replication initiator protein, and which contributes to the inactivation of DnaA. An excess of datA sites provided on plasmids has been found to lead to both a delay in initiation of replication and in cell division during exponential growth. Here we have investigated the effect of datA on the cell division block that occurs upon inhibition of replication initiation in a dnaC2 mutant. We found that this checkpoint mechanism was aided by the presence of datA. In cells where datA was deleted or an excess of DnaA was provided, cell division occurred in the absence of replication and anucleate cells were formed. This finding indicates that loss of datA and/or excess of DnaA protein promote cell division. This conclusion was supported by the finding that the lethality of the division-compromised mutants ftsZ84 and ftsI23 was suppressed by deletion of datA, at the lowest non-permissive temperature. We propose that the cell division block that occurs upon inhibition of DNA replication is, at least in part, due to a drop in the concentration of the ATP-DnaA protein.

Autoregulation of the Escherichia coli replication initiator protein, DnaA, is indirect.

The expression of dnaA is autoregulated, in that transcription of the gene increases when DnaA is inactivated (and initiation of replication prevented) and decreases when DnaA is supplied in excess. However, the inactivation of DnaA does not necessarily lead to increased DnaA production, as dnaA(Ts; temperature sensitive) strains which are integratively suppressed by derivatives of the plasmid R1 do not show temperature-induced derepression. Several possible explanations for this unanticipated behaviour were considered and ruled out. We suggest here that the completion of a critical step in initiation may prevent dnaA derepression: although DnaA would be required to complete this step at oriC, DnaA(Ts) would be sufficient at the R1 origin. Autoregulation of dnaA has been attributed to the binding of DnaA at a consensus binding site in the dnaA promoter region. We show here, using reporter systems, that this DnaA-binding site is not required for the autoregulatory response. We find, further, that replacement of the chromosomal dnaA gene with one containing a mutated binding site causes no demonstrable phenotypic change: cells with the mutant gene show no disadvantage in competition with dnaA+ cells.

Transcriptional activation of the origin of coliphage λ DNA replication is regulated by the host DnaA initiator function

The initiator of phage λ DNA replication, the λ O protein, is considered to be an analogue of the initiator of DNA replication (DnaA) of its host, Escherichia coli. Both specifically recognize their origins of replication, oriλ and oriC, respectively, and organize the assembly of specific replication complexes. However, DnaA has an additional activation function, acting on oriC-proximal DnaA-boxes, and regulating transcription initiated at promoters in and around oriC. Here, we demonstrate that λ plasmid replication can be synchronized by a temperature shift-down that caused renaturation of the previously denatured DnaAts protein. Moreover, we show that elimination of the activating DnaA function affects transcriptional activation at oriλ. DnaA may act by binding to DnaA-boxes, situated around the λ p R promoter; there are no such sequences in oriλ. Our results begin to explain in molecular terms why λ plasmid replication is DnaA-dependent [Kur et al., J. Mol. Biol. 198 (1987) 203–210] and why the initiation of phage λ DNA replication is blocked (in E. coli devoid of prophage Rac) after inactivation of DnaA [Wggrzyn et al., Genetics (1995) in press]

Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli

The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA+ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA+ gene was expressed in the presence of the inducer isopropyl-1-thio-beta-D-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30 degrees C, or after inactivation of DnaA by a shift to 42 degrees C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom- plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately.(ABSTRACT TRUNCATED AT 250 WORDS)