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

The goal of my PhD research was to develop and use single-molecule tools to understand the molecular mechanisms in DNA replication. To this end, we developed a new fluorescence imaging technique, by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. Next, we visualised the exchange behaviour of two proteins within the E. coli replication system under physiologically relevant protein concentrations: polymerases and SSB. We see rapid exchange, depending on the concentration of competing protein in solution. The emergence of the concentration dependence illustrates the importance of studying the molecular sociology of multi-protein complexes at the single-molecule level. Then we aimed to determine the effect of the E. coli RarA protein on DNA replication and repair. Combining in vitro and in vivo techniques, we propose that RarA activity is involved in the creation of gaps in lesion-containing DNA templates, and thereby commits the cell to the translesion DNA synthesis repair pathway. Finally we describe the first single-molecule experiments done on the reconstituted S. cerevisiae replisome, characterising the kinetics of leading-strand synthesis. We confirm a previously reported observation that the MTC complex enhances the speed of the replication fork by 2 fold. Surprisingly, however, our data suggest that MTC only transiently interacts with the replisome through a weak interaction.