At low forces (~3 pN) in a flow of buffer, single-stranded (ss)DNA forms  compact structures while double-stranded (ds)DNA is essentially fully extended. This property can be exploited in a simple DNA tethered-bead assay to observe inter-conversion between ss and dsDNA during DNA replication of single molecules in real time. With multiple observations, we are able to quantify parameters including lifetimes of active replisomes, their rates of DNA synthesis, the extent of DNA elongation during single binding events (processivity), their pausing while protein complexes exchange from solution, and the efficiency and consequences of their blockage at replication barriers.  Two studies that exploit the tethered-bead assay will be described to illustrate the power of this technique.

In the first, we discovered a new-transient protein-protein interaction in the E. coli replisome, between the epsilon proofreading subunit of DNA Pol III and the processivity-determining sliding clamp. This allows us to model the structure of the active replicase complex, and how it changes during proofreading and when it encounters a template lesion. In the second system, we show that the speed of DNA synthesis is inversely correlated with the efficiency of replication fork blockage on single DNA molecules during replication termination.

In the future, use of proteins tagged with fluorophores in single-molecule DNA replication assays, coupled with total internal reflection fluorescence (TIRF) microscopy will provide a powerful tool to correlate protein exchange and conformational changes in the replisome with events like transient pauses, replication blocks, lesion bypass and replication restart.