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. Author manuscript; available in PMC: 2017 Mar 27.
Published in final edited form as: J Mol Biol. 2016 Feb 22;428(6):1053–1067. doi: 10.1016/j.jmb.2016.02.017

Figure 4. A monomer of Pif1 can unwind dsDNA in a single turnover. a).

Figure 4

Stopped-flow FRET-based unwinding by 15 nM Pif1238–859 of a substrate (20 nM) with a 23 bp dsDNA, a 5′-dT10 and a 22 nt 3′-tail of mixed sequence composition. The reactions were started by addition of either 0.5 mM ATP (black) or 0.5 mM ATP + 3.5x of the r.a. TRAP (red) or 0.5 mM ATP + 3.5x of the r.a. TRAP + 250 nM dT60 as protein trap (blue). b) Lag times as function of dsDNA length for experiments performed in the absence (black) or the presence of the r.a. TRAP (red) or the r.a TRAP + dT60 (blue). c) ATP concentration dependence (500 μM, black; 250 μM, gray; 125 μM, red; 50 μM, blue) of unwinding by Pif1238–859 using the same substrate as in a). d) Rate of unwinding as a function of ATP concentration determined using different lengths of the dsDNA for Pif1238–859 (black) and full-length Pif1 (gray). e,f) Same experiments as in a) and c) using full-length Pif1.