Figure 1.

Optical-tweezers assay to monitor T7 DNAp activity. (A) Schematic of the experimental layout. A DNA molecule with a 5′ overhang (right) is held between two optically trapped microspheres. In the presence of T7 DNAp and nucleotides, activity is measured by determining DNA length changes while clamping the force. (B) Force-extension curves of dsDNA and ssDNA. At tensions above 6 pN, polymerization by DNAp at constant tension results in shortening of the DNA construct, whereas exonucleolysis leads to lengthening (dashed arrows). (C) Typical time trace of T7 DNAp activity at a force of 45 pN, with six different behaviors indicated. (D) Schematic of observed behaviors: polymerase, exonucleolysis, four different types of pauses, and two direct switches in activity. In these diagrams, solid lines between circles represent current behavior, and dashed lines indicate preceding and subsequent behaviors. The axes are equivalent to those in (C).