Unwinding of RNA/DNA duplexes by wild-type and Has1p mutants. (A) Time course for ATP-dependent unwinding of 3′ duplex and 5′ duplex by wild-type Has1p. In short, 50 nM duplex was incubated with 50 nM protein with or without 1 mM ATP, at 30°C for the time indicated in minutes. To prevent reannealing of the displaced 32P-labeled oligonucleotide, 1 μM cold DNA oligonucleotide was added as a competitor. Products were separated on a 15% polyacrylamide gel, which was then subjected to autoradiography. (B) Unwinding activities of the wild-type and Has1p mutants. An aliquot of 50 nM duplex (3′ top panel, 5′ bottom panel) was incubated either with 50 nM protein (molar ratio 1:1, as indicated) or 10 nM protein (molar ratio 1:5, as indicated). The reactions were carried out for 60 min at 30°C in the presence of 3 mM ATP. To prevent reannealing of the displaced 32P-labeled oligonucleotide, 1 μM cold DNA oligonucleotide was added as a competitor. Products were separated on a 15% polyacrylamide gel, which was then subjected to autoradiography. As control, the duplex was either heat-denatured for 2 min at 95°C, quick-cooled on ice and then loaded, or starting material (SM) containing the same amount of duplex was loaded. (C) Quantification of the experiments that were performed with 50 nM duplex and 50 nM enzyme, and incubated for 60 min at 30°C in the presence of 3 mM ATP. The standard errors of estimation were derived from the curve fits based on three independent sets of experiments.