Single-molecule studies of the replisome Visualisation of protein dynamics in multi-protein complexes

DNA replication, or the duplication of parental double-stranded DNA (dsDNA) into a pair of identical copies, is essential to the transmission of hereditary information from cell to cell and thus the propagation of all life. It is a fundamental cellular process that is carried out by a multi-protein complex known as the replisome. Since the identification of the first replication proteins by Arthur Kornberg in the 1950s, ensemble-averaging biochemical techniques have been successfully used to study the roles of the various proteins within the replisome. However, the coordination of the multiple activities within the replisome involves transient intermediates and dynamic conformational changes that are difficult, if not impossible, to observe with ensemble experiments. Recently, new single-molecule techniques have been developed to study the dynamics of proteins with a high precision and without the need for population averaging. This thesis centers on the use of these approaches to study the dynamic behaviour of the replisome.