Figure 1.

Effect of HNE on recombinant WRN. (A) HNE covalently modifies WRN in a concentration-dependent manner. Purified WRN in the presence of BSA was incubated with increasing molar ratios of HNE for 10 min at 30°C. Samples were boiled in SDS loading buffer, subjected to SDS-PAGE, blotted and probed for HNE followed by stripping and reprobing for anti-WRN antibodies. (B) Fold increase in WRN modification from the data shown in (A). (C) Effect of HNE modification on WRN helicase activity. Purified WRN was incubated with increasing molar ratios of HNE and enzymatic assays were performed as described in Materials and Methods. (D) Percent of control helicase activity from the data shown in (C). (E) Effect of HNE modification on WRN exonuclease activity. Purified WRN was incubated with increasing molar ratios of HNE, and enzymatic assays were performed. (F) Percent of control exonuclease activity from the data shown in (E). (G) Effect of HNE modification on WRN ATPase activity. (H) Percent of control ATPase activity from the data shown in (G). Mock, control enzymatic activity performed with WRN that has been treated as HNE modified WRN but without HNE. Lane 14, heat denatured helicase substrate. All data bars are the mean of at least three independent experiments with SDs indicated by error bars. Statistical analysis occurred via one-way analysis of variance with Tukey's multiple-comparison test (*P < 0.05; ***P <0.001).