The initiation of DNA replication in Echerichia coli is a tightly regulated event; a phenomenon mediated by many factors. One of the most important of these are the DnaA protein in that it function as the iniator; the trans-acting substance in Jacobs replicon theory. The DnaA protein initiates replication by binding to its respective binding sites, the DnaA boxes, in the origin, oriC, creating what is known as the initial complex. The ATP-bound form of DnaA is in able to bind to additional sites known as ATP-DnaA boxes, this results in the unwinding of an AT-rich region located at the left border of oriC, creating the open complex. In the subsequent steps, the DnaA protein aids in the loading of the DnaB helicase which is followed by the assembly of the replication machinery. The cell utilizes several strategies to inhibit re-initiation of a newly initiated origin and among these are the titration of DnaA to other sites on the chromosome, the inactivation of ATP-DnaA by the hydrolyzation of ATP to ADP and the sequestration of OriC by the binding of the SeqA protein.
Given the fact that the initiation event is crucial in cell cycle control in bacteria, a better understanding of the factors involved is needed to fully appreciate the extent of this process. To date efforts have been made to study the localization of DnaA in the cell and to find possible foci using immunostaining (Newman and Crooke, 2000), but the conclusion has been that for a better understanding of subcellular localization of DnaA during the cell cycle, a DnaA-GFP fusion will be needed. This could make it possible to observe the initiation event under the light microscope. This has proved to be easier said than done. Other labs have tried fusing the GFP protein to both the N-terminal and the C-terminal of the DnaA protein, two strategies which produced toxic proteins; this effect could be explained by the sheer size of the GFP Ò-can structure ¡V that it interfered with oligomerization when bound to the N-terminal and with DNA-binding when bound to the C-terminal. Our strategy to produce a non-toxic DnaA-GFP fusion has been to exploit the fact that the DnaA is built up of four sub-domains with different functions. We chose two strategies; in the first method, we wanted to insert the GFP-mut2 protein into the middle of the second domain of DnaA thus localizing the Ò-can of the GFP-mut2 protein to the variable region, where it might not interfere with the normal function of DnaA. In the second strategy, we wanted to produce a protein consisting of the first two domains of DnaA with GFP-mut2 fused to the C-terminal part of domain II. The first strategy, the insertion of GFP-mut2 into the middle of domain II, failed because the SOEing method employed to create the recombinant protein, did not handle larger constructs very well. We succesfully created the recombinant gene consisting of gfp-mut2 fused to the first two domains of dnaA. We had, however, problems in expressing the DnaA(I/II)-GFP-mut2 protein in vivo; GFP was clearly present as the cells fluoresced, but western blot analysis failed to give evidence for the expression of DnaA(I/II)-Gfp-mut2. Sequencing showed that the cause of the expression problem was the deletion of the dnaA(I/II) part of the recombinant gene, a mutation which had arisen in vivo, most likely as a result of some recombinatorial event. As the work on this thesis progressed, we learned that the Crooke group had succesfully created a GFP tagged DnaA protein, inserting the eGFP gene into the middle of domain II. We analyzed the strain carrying the DnaA-eGFP fusion protein, and observed a variable number of fluorescent foci. However, further studies are needed to make any inferences concerning the interpretations of these.