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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: Methods. 2016 May 9;108:65–78. doi: 10.1016/j.ymeth.2016.05.003

Figure 5. One-pot assay to measure the chemical step size (bp/NTP hydrolyzed) of the helicase-DNAP catalyzing leading strand DNA synthesis.

Figure 5

(A) One-pot assay to measure the base pairs unwound-copied by the T7 helicase-DNAP and dNTP hydrolyzed by the helicase using [α32P]dNTPs, which quantify the kinetics of dNTP hydrolysis to dNDP and the incorporation of dNMP into the DNA. (B) Thin-layer chromatography (PEI-cellulose) shows the separation of [α32P]dNTPs from the corresponding [α32P]dNDPs and DNA (labeled with [α32P]dNMPs). (C) Kinetics of total dNDPs production and total dNMPs incorporation into the DNA by the helicase-DNAP on the 100% AT-rich fork DNA substrate. (D) Kinetics of total dNDPs production and total dNMPs incorporated on the 50% GC-rich DNA substrate.