Single Molecule Fluorescence In-vivo

J-F Allemand recently started a collaboration with the group of B. Michel from the CGM in Gif/Yvette. In collaboration with G. Lia from this group he implemented in the LPS a single molecule in vivo technique, named detection by localization, in order to monitor replication forks in E. coli. The key point of this technique is to use fluorescently tagged proteins in living bacteria. When the proteins are freeely diffusing in the cytoplasm, due to the relatively long integration time of the camera, fluorescent proteins appear as a backgroung signal. But when these proteins are interacting with a slowly diffusing component, like the cell membrane or in our case the machinery responsible for DNA replication (the replisome), all the photons emitted during the integration time of the camera are coming from the same spatial origin and the tagged protein appear as a bright spot. The intensity of the spot is quantitative, thus the intensity of this spot gives the number of proteins that are present. By using this technique we could monitor the exact composition of the replisome in real time.  This allowed our collaboration to confirm that despite they are only 2 strands of DNA to replicate 3 polymerases are present in a replisome. Importantly, since we improved our technique to have access to the dynamics of the replisome composition, it was demonstrated that the 3 polymerases are not always the same at the replisome. There is an exchange with proteins from the cellular pool every ~1s in our experimental conditions. By monitoring the coordination of this exchange with another protein present at the replisome, indicative of the presence of single stranded DNA in the cell, the group demonstrated that the polymerase that is exchanged is the one responsible for the lagging strand synthesis. This strand is the one that is not synthesized continuously, its synthesis is performed in fragments, named the Okazaki fragments. Analysis of the synchrony of cellular elements imply that a polymerase is exchanged for every fragment. Consequently is could be proposed that the third polymerase present in the replisome can be used when there is a delay in the binding of a new polymerase, as a spare wheel in car that can be used if there is a problem on an active tyre. More recently this collaboration started to monitor the evolution of the replisome when this cellular machinery is blocked. The motivation for that is that some cellular processes can stop the progression of the replisome during replication and the repair of this stall leads to genomic instabilities, that are then important to understand. For the moment our group is using an articial stalling process : a temperature sensitive helicase. Helicases are molecular motors that unwind the double helix (see the micromanipulation part). In a strain where the replicative stops when the temperature is about 42°C we observe how the different components of the replisome leave the stalled fork and when, and how many, components are coming to repair the system. Our main observation is that replisome is dismounted piece by piece and that one of the 3 polymerases is more stable and that an enzyme active in the repair of other cellular processes (RecA) has an active role is this repair.

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