In vivo dynamics of DnaA and its regulators

An important step during the bacterial cell cycle is the duplication of the chromosome. This key process has to be tightly regulated to ensure that the number of chromosomes per cell remains constant. Chromosomal DNA replication is initiated at the origin of replication by the highly conserved initiator protein DnaA. DnaA binds to specific DNA sequences at the oriC region and initiates replication by forming a stable, helical right-handed nucleoprotein complex, which leads to local unwinding of the double-stranded DNA. To prevent untimely and overinitiation the initiation step is highly regulated. In B. subtilis, YabA and Soj (ParA) play major roles in regulating the initiation of replication. YabA functions as an anti-cooperativity factor of DnaA and furthermore tethers DnaA to the replication machinery. Soj, on the other hand, was shown to stimulate DNA replication in its dimeric form and to inhibit DnaA helix formation as a monomer. This study reveals that the localization of YabA with respect to the origin of replication and the replication machinery is similar to the pattern previously observed for DnaA, indicating that YabA can localize at the origin of replication but that it is primarily localized at the replisome. By using in vivo fluorescence microscopy after photobleaching (FRAP), I could show that the exchange rates of DnaA molecules bound to the origin of replication or at the replication machinery are similar and in the lower second range. Furthermore, a deletion of yabA or soj- spo0J significantly decreased the turnover of DnaA. In addition, YabA was shown to have similar recovery half-times as DnaA. As a second approach single molecule microscopy in live cells was applied. The study of DnaA at the single molecule level reveals that DnaA is indeed a highly mobile protein and does not rest for more than a few hundred milliseconds at its individual binding sites. Because DnaA also acts as transcription factor at several sites throughout the chromosome, these sites also include several promotor sites, and not only oriC or the replication forks. Furthermore, a deletion of soj-spo0J increased the residence time of DnaA molecules, which is in accordance with results obtained in FRAP experiments. On the contrary, a DnaA variant carrying a single amino acid substitution, which renders it impaired in ATPase activity and leads to an underinitiation of replication, displays an even shorter residence time than wild type DnaA. Therefore, even mild differences in residence time can lead to aberrant initiation frequencies. YabA molecules, on the other hand, tend to be significantly more static, indicating that DnaA and YabA are not scanning together over the chromosome. Strikingly, E. coli DnaA showed an oscillation of DnaA molecules between the two cell halves and a diffusion constant and residence time similar to B. subtilis DnaA, revealing that DnaA is highly dynamic in two different bacterial species. Subsequently, the observation that DnaA in E. coli oscillates might support the notion of a mechanism in which DnaA is chased by a slower regulatory protein that upon interaction induces the release of DnaA from its binding ixsite, thereby inducing the alternating pattern of DnaA. Fast turnover rates may be advantageous to integrate many regulatory inputs into the decision whether to initiate or not. In B. subtilis the two regulators YabA and Soj could act in part through a stimulation of DnaA turnover at oriC and the replication machinery, thereby regulating the initiation of replication.