Background: The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF. The dnaA gene encodes the DnaA protein, which is required for the initiation of DNA replication from oriC. The dnaN gene encodes the ß-subunit of DNA polymerase III holoenzyme (Pol III), which acts as a clamp that tethers the holoenzyme to the DNA template during replication. The RecF protein, encoded by recF, is required for the resumption of DNA replication after UV damage. In this study, we are examining whether an increase of the intracellular concentrations of DnaA, ß-subunit, and RecF together and separately cause changes in the rate of initiation, the rate of fork movement, the length of the C period of the cell cycle, and in cell viability.
     Methods: Plasmids have been constructed that contain all combinations of the dnaA, dnaN, and recF genes under the control of an IPTG-inducible promoter. Cells that have been grown in the presence of IPTG at various concentrations to induce the synthesis of these proteins were examined by Western analysis, flow cytometry, and pulse labeling with ³H-thymidine. Flow cytometry reveals whether chromosomal origins initiate at the correct time during the cell cycle and if forks move to the terminus. The C period was determined by inhibiting initiation with rifampicin and following the decrease in the rate of DNA synthesis. The SOS response was measured using a strain containing a sulA::lacZ fusion.
     Results:  Western and flow cytometry results indicate that cells with levels of DnaA four-fold higher than wild-type contain partially replicated chromosomes at the end of three hours of rifampicin treatment. Replication fork movement is slower in these cells at this higher induction level. Cell viability also decreased, but the SOS response is not induced. Cells with increased amounts of RecF alone appear to be normal with respect to initiation timing and replication fork movement, as are cells with increased levels of both DnaA and RecF together. A mutation in the recF gene, however, further decreases viability of cells over-expressing DnaA.
     Conclusions: It appears that a concomitant increase of RecF protein helps overcome the detrimental effects caused by an increase in the number of replication forks and a decrease in replication fork movement observed when the concentration of DnaA is raised four-fold.
     Acknowledgments: Funding for this project has been provided by the National Science Foundation, USA, grant MCB-9507209 to J.W.Z.