DNA constructs were immobilized on polyethylene glycol (PEG; Laysan Bio Inc.)–functionalized quartz surface to minimize nonspecific protein binding onto the surface. Immobilization was held by neutravidin (Pierce) and biotin interaction at the ends of DNA and biotin-modified PEG on the surface. Approximately 10 to 50 pM concentrations of DNA constructs were injected into the imaging chamber to achieve an appropriate density for single-molecule imaging. An oxygen scavenger system with catalase, glucose oxidase, and d-glucose was used to prevent rapid photobleaching of fluorescent dyes. The reaction buffer contained 50 mM tris-HCl (pH 7.5), 10 mM MgCl2, bovine serum albumin (50 μg/ml), 1 mM dithiothreitol, Trolox (1 mg/ml) (Sigma-Aldrich), and the oxygen scavenging system of glucose oxidase (1 mg/ml) (Sigma-Aldrich) and 0.4% (w/v) d-glucose (Sigma-Aldrich). A flow channel was constructed by assembling a coverslip onto a microscope slide using double-sided tape and sealing the edge of the slide with epoxy. A 1-ml syringe was connected to the outlet hole on the flow chamber through tubing, and a pipette tip reservoir containing a reaction solution was built on the inlet hole. When the syringe was pulled, the solution was delivered into the chamber at a rate of ~10 μl/s. The multiturnover reaction was started by introducing a buffer containing ExoIII and Mg2+ into DNA substrates tethered on the surface of an imaging chamber at room temperature. In contrast, the single-turnover reaction, where DNA substrates were preincubated with ExoIII for ~2 min in the absence of Mg2+, was started by flowing in Mg2+ buffer.

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