Dynamics of the bacterial replisome:Biochemical and single-molecule studies of the replicative helicase in Escherichia coli

All the instructions that regulate all aspects of the life cycle of any living cell on Earth are safely stored in a molecule called DNA. It is therefore a fundamental process for life that the information on the DNA is preserved and faithfully transmitted to the next generation. The division of a cell into two daughter cells, however, requires that an identical copy of the DNA is synthesized. For this purpose, cells employ a multi-protein machinery to guarantee error-free duplication of the DNA, process called DNA replication. This thesis investigates the finely tuned coordination between the various components of the DNA-replication machinery in Escherichia coli exploiting the specificity of biochemical ensemble-averaging approaches to probe interactions and the kinetic resolution of single-molecule imaging. In particular, the dynamics of the interaction between the replicative DnaB helicase and the DNA polymerase Pol III holoenzyme is described and the implications on the replication mechanisms are presented. Data are provided to argue that E. coli has evolved regulatory mechanisms that confer to the duplication machinery flexibility and the ability to adapt. The robustness and the reliability of DNA duplication are a consequence of this plasticity, with the large number of weak interactions within the duplication machinery supporting great stability, but also allowing exchange of protein components