Abstract
E.coli DNA replication is a complex process that requires the coordinated action of the many protein subunits and co-factors that constitute the replisome. Single-molecule assays capturing replisome activity in vitro are thus far mainly limited to low-resolution flow stretching assays (100s of nm), leaving much of the detailed system dynamics obscured by noise. Here we directly observe the activity of E.coli replisome components such as Pol III DNA polymerase and DnaB helicase at the much higher resolution that is achievable with magnetic tweezers (MT, ∼5 nm). As the replisome requires temperatures above 30°C to display sufficient activity, all experiments were performed at 36°C using a home-built temperature control system. Quantitative analysis of large datasets obtained through the MT multiplexing capacity reveal detailed pause kinetics of pol III DNA polymerase during strand displacement synthesis and DnaB helicase unwinding activity.
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