CPM Seminar

Single-molecule studies of DNA replication in live cells

Rodrigo Reyes

Department of Biology
McGill University

DNA replication requires the coordinated activity of numerous factors which associate in a multi-protein machine, the replisome. In Escherichia coli, a pair of replisomes are loaded at a single point of the circular chromosome and proceed in opposite directions until synthesis is completed, a process that takes about 1 hour. It has been assumed that replisomes are stable molecular assemblies, nevertheless this is not supported by measurements done in vivo. Using single-molecule microscopy in live cells we have determined the binding kinetics of individual replisome subunits in active replisomes. We have also obtained stoichiometries per replisome and copy numbers of individual components in the cell, allowing us to test mechanistic models on their mode of action. Our data demonstrates that the bacterial replisome is both stable and flexible, likely to facilitate processing of the frequent obstacles that replisome encounters on DNA. Current efforts in our lab aim to obtain quantitative information from microscopy in budding yeast, a model for eukaryotic organisms, to test the evolutionary conservation of similar mechanism in DNA replication.