In vitro & in vivo single molecule approaches to DNA replication
Jean-François Allemand (École normale supérieure)
DNA replication is an essential step of the cell cycle. Despite many biochemical studies, physical approaches can still bring new informations on some process of DNA replication.
In vitro, using micromanipulation tools, we have shown that force can trigger some DNA polymerases into an exonuclease mode where, instead of synthesizing DNA, the polymerase goes backward and removes nucleotides. This can lead to a mechanical method for DNA sequencing.
In vivo, in E. coli, using single molecule fluorescence techniques we have quantified the real time composition of the replisome, the machinary that copies DNA. We observed a time variation of the composition of the replisome with a number of DNA polymerases that varies from 2 to 3. We will see how these fluctuations are related to the synthesis of the so called Okazaki fragments. We will describe what happens to this scenario when the replication is blocked under specific conditions.