Advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. In a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important new direction is the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies.
We are applying a single-molecule approach to study DNA replication, a process that is supported by a large, multi-protein complex containing a number of different activities. I will present recent results of single-molecule studies of replication in bacterial and eukaryotic systems, both in vitro and in vivo. By combining the mechanical stretching of individual DNA molecules with the fluorescence observation of individual proteins, we visualize the dynamic behavior of phage and bacterial replication complexes during replication in vitro. Further, I will present data from single-molecule replication studies in X. laevis oocyte extracts. We have developed a novel imaging scheme that permits single-molecule fluorescence experiments at concentrations of labeled protein that were hitherto inaccessible. Using this method, we visualize, in real time, origin firing and fork movement. Finally, I will present imaging studies that aim to visualize in live cells the interplay between DNA replication and repair at the single-molecule level.