ABSTRACT
The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, β clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, β clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the β clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 μm) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional ∼100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.
Overexpression of the Replicative Helicase in Escherichia coli Inhibits Replication Initiation and Replication Fork Reloading
