Figure 3. Requirement for ATP hydrolysis and a conserved lysine residue in CeWRN-1 for helicase activity.

(A) Effect of ATP and ATPγS on CeWRN-1 helicase activity. Reaction mixtures contained 1 nM substrate, 30 nM CeWRN-1, and 2 mM ATP (or 2 mM ATPγS) as indicated. Helicase reactions were carried out as described in Materials and Methods. The reaction products were visualized by autoradiography: lane 1, no enzyme control; lane 2, no nucleotide control; lane 3, in the presence of ATP; lane 4, in the presence of ATPγS; and lane 5, heat-denatured DNA substrate control. The substrate and product are shown at the right. (B) ATPase activity of CeWRN-1 in the presence of the indicated DNA effectors. Reaction mixtures contained 20 nM CeWRN-1 (white symbols) or 20 nM CeWRN-1-K255A (black symbols), 2 mM ATP, and 25 μg/mL DNA effector, and reactions were carried out at 37 °C for the indicated times. The amount of inorganic phosphate (P_i) released by ATP hydrolysis was determined as described in Materials and Methods: (△ and ▲) supercoiled plasmid, (○ and ●) blunt-ended linear dsDNA, (□ and ■) circular M13mp18 ssDNA, and (◇ and ◆) no DNA. (C) Comparative helicase assay for wild-type CeWRN-1 and CeWRN-1-K255A. Reactions were performed with 1 nM DNA substrate and varying amounts of purified wild-type CeWRN-1 (lanes 1–7) and mutant CeWRN-1-K255A (lanes 8–14) at 37 °C for 15 min. The reaction products were analyzed by 10% neutral PAGE. Radiolabeled DNAs were visualized by autoradiography: lanes 1 and 8, no enzyme control; and lanes 7 and 14, heat-denatured DNA substrate control. Lanes 2–6 show wild-type CeWRN-1 protein titration (2.2, 4.4, 8.8, 17.6, and 35.3 nM, respectively), and lanes 9–13 show CeWRN-1-K255A protein titration (3.1, 6.2, 12.5, 25, and 50 nM, respectively). The substrate and product are indicated schematically at the right.