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The initiation of replication – only once, please

DNA replication is a critical event in the cell division process. The genetic material must only be replicated once. So, how does a bacterial cell ensure that only one single replication occurs and that the process is not repeated several times? Microbiologists under the leadership of Prof. Dr. Peter Graumann from the Institute of Biology II in Freiburg, in cooperation with international cooperation partners from Paris, have deciphered a mechanism in the bactericum Bacillus subtilis that involves the capture of a specific protein.

Bacillus subtilis under the light microscope. Several dozen small rods can be discerned © Wikipedia

Bacteria such as Bacillus subtilis replicate their genome under normal conditions within an hour. During the cell division process, the daughter cell must receive no more and no less  than one complete chromosome. Bacteria that undergo repeated replication slow down to the extent that some of them die. It is assumed that the protein DnaA, which is regarded as the initiator of replication, plays a key role in this process. Only when this protein has attached to a certain DNA stretch, which is known as the origin of replication, does the DNA double helix open up and unwind at the area in question. This leads to what is known a replication fork in which the replication machinery starts the copying process. "How does a bacterial cell prevent DnaA from attaching to the origin of replication more than once per cell cycle, thus inducing further replication," asks Prof. Dr. Peter Graumann, head of a group of researchers working in the Department of Microbiology at the Faculty of Biology, University of Freiburg.

Pulling away

Last year, Graumann’s team and their French cooperation partners carried out co-localisation experiments in order to clarify this issue. Their research was based on the observation that the initiation of the copying process becomes confused when the protein YabA is missing. The bacteria start to replicate over and over again when YabA is absent. Does the protein affect the binding of DnaA to the origin of replication and hence the start of the copying process? And if so, how? The researchers coupled the genes for YabA, DnaA and another protein of the replication machinery (DnaX) with different genes encoding proteins of the GFP (green fluorescent protein) family. The proteins then fluoresced different colours in the cell. Under the fluorescence microscope, the biologists were then able to discern the location of the molecules during the replication and cell division process.

The photo shows four pictures – origin-CFP, DnaA-YFP, DnaX-RFG and overlay, including three photos showing Bacillus subtilis cells under the fluorescence microscope. The bars indicate the dividing walls between the cells. The left photo shows the coupling of the origin areas with CFP; the green glow shows the location of the areas at the cell poles. The second photo shows the location of DnaA in the centre of the cells, coupled with red fluorescing YFP. The third photo shows the components of the replication machinery in yellow. The photo on the right shows an overlay of the three photos. © Prof. Dr. Peter Graumann

The glow revealed that both DnaA and YabA remained in the centre of a bacterial cell during replication. The replication machinery is also located in the centre. The newly replicated DNA is pulled past this complex and the newly replicated DNA stretches move towards the cell poles. "The mechanism through which the initiation of replication is regulated is a ‘pulling-away mechanism'," said Graumann. Only at the beginning of replication are the areas of origin found in the same cell region as the replication machinery and the DnaA initiator protein. The origin areas subsequently separate spatially from the replication machinery and the DnaA initiator protein. What causes this to happen? YabA has a binding site for DnaA and the replication machinery. Does it retain the initiator protein in order to prevent it from reattaching to the origin region after a one-time replication initiation? This is in fact what experiments with Bacillus strains and without YabA suggested. In YabA-deficient strains, the DnaA protein does not remain in the centre of the cell, but instead is found all over the cell interior.

Open questions

"YabA works like an adapter," said Graumann. "It couples DnaA to a component of the replication machinery, thereby arresting the protein." It is still unclear how DnaA detaches from the origin, but Graumann and his team are currently working on the clarification of this point. They assume that another protein, Soj, might be involved as it also appears to play a role in the regulation of initiation. If Soj is not present, then the cell does not know how many replication rounds it has gone through. "Its function might be to remove DnaA following the initiation of DNA," speculates Graumann. "But further experiments are needed to clarify this."

Another question is: how does DnaA initiate replication? Although it is known that DnaA does initiate replication, the scientific question as to how it does this remains unanswered. Graumann and his team are keeping their eye on the ball as far as this question is concerned. Their experiments on molecular replication mechanisms and their regulation in bacteria such as Bacillus subtilis are of great general importance for two reasons: “On the one hand, it is a basic biological process,” said Graumann. “And this, in itself, is of major interest.” On the other hand, the structure and function of DnaA resembles that of known eukaryotic initiator proteins. Therefore, a detailed understanding of the processes at the replication fork in Bacillus subtilis might at some point in the future also provide valuable insights into the replication mechanism of higher organisms. And this could prove to be of major importance for cancer research, as some cancer cells are known to be unable to correctly regulate the number of replications per cell division.

Further information:

Prof. Dr. Peter Graumann
Institute of Microbiology
Schänzlestr. 1
Tel.: ++49-(0)761/203-2630
Fax: ++49-(0)761/203-2773
E-mail: peter.graumann(at)biologie.uni-freiburg.de

Website address: https://www.biooekonomie-bw.de/en/articles/news/the-initiation-of-replication-only-once-please