Single-molecule Observations of Replisome Structure and Function

Abstract

DNA replication requires the coordinated activity of a large number of enzymes at the replication fork. Understanding the mechanisms controlling this organization requires a direct probing of the dynamics of fully functional replisomes during replication. Observations at the single-molecule level provide the most direct way to visualize the complex biochemistry of the replisome and to quantify the many transient intermediates essential to replication. We present a novel assay that combines the observation of individual fluorescently labeled proteins with the mechanical manipulation of DNA. Surface-tethered DNAs labeled with quantum dots are hydrodynamically stretched and imaged with a TIRF microscope. Activity of the replisome is observed as a change in the DNA length due to the differing force-dependent extension of single- and double-stranded DNA at low pico-Newton forces. We employ a two-color imaging scheme to monitor DNA length in real-time and to stroboscopically image fluorescently labeled single proteins interacting with DNA. Observation of labeled proteins in an ongoing replication reaction allows us to pose structural questions about the stoichiometry and exchange of proteins at the prokaryotic replication fork. We will discuss preliminary results on primer extension by the T7 DNA polymerase and strand-displacement synthesis by the coupled activity of the T7 helicase and polymerase.