Single-Molecule Studies of DNA Replication: The Plasticity of Multi-Protein Complexes

Abstract

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 and held together by protein-protein and protein-DNA interactions of many different strengths. While the classical textbook picture of the replisome is one in which the protein components, in particular DNA polymerases, are efficiently retained and re-used for a large number of cycles of Okazaki-fragment synthesis on the lagging strand, our single-molecule studies imply a very different picture. I will present fluorescence-based single-molecule imaging experiments, in vitro and in vivo, of the E. coli DNA-replication machinery and demonstrate that new DNA polymerases are recruited by the replisome on a timescale of mere seconds. Further, simultaneous observation of both leading- and lagging-strand synthesis in a single replisome suggests that the replication machinery employs a number of different molecular mechanisms to achieve coordination between the DNA polymerases on each strand.