Real-time single-molecule observation of rolling-circle DNA replication

Abstract

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.

Figure 1.

(a) Schematic of single-molecule rolling-circle assay. ‘SA’, streptavidin. Upon leading-strand synthesis, DNA is displaced from the circle as the replisome ‘rolls’ around the template. The emerging ‘tail’ is converted to dsDNA via lagging-strand synthesis. As a result, the DNA that couples the M13 circle to the surface increases in length and is extended in the direction of flow. (b) Example field of view. Note both the length and number of products. Each flow cell has thousands of such fields, allowing for large numbers of products to be observed in a single experiment.

Figure 2.

Kymographs of example DNA molecules from (a) T7 and (b) E. coli replication experiments. Endpoint trajectories are plotted vs. time to obtain rates of synthesis by fitting with linear regression (c). Rates of shown traces are: 99.4 bp s⁻¹ (T7) and 467.1 bp s⁻¹ (E. coli).

Figure 3.

(a) Length distributions of replication products, with means of 25.3 ± 1.7 kbp (T7) and 85.3 ± 6.1 kbp (E. coli). (b) Rate distributions of single molecules, with means of 75.9 ± 4.8 bp s⁻¹ (T7) and 535.5 ± 39 bp s⁻¹ (E. coli). The log of the rates are plotted to allow simultaneous display of both broad distributions.

Figure 4.

(a) Rates of T7 DNA synthesis at various ddGTP concentrations. (b) Lengths of T7 replication products at various ddGTP concentrations. Values are means and error bars represent standard error of the mean.