Research Paper Volume 4, Issue 6 pp 417—429
DNA binding residues in the RQC domain of Werner protein are critical for its catalytic activities
Figure 3. Mutations in RQC domain of WRN result in significant decrease in DNA unwinding, DNA binding and ATPase activity. (A) DNA unwinding activity of WRN variants on forked duplex substrate. 1, 2, 5 nM WRN wild type (lane 2 to 4) or WRN RQC variants (lane 5 to 7, R993A; lane 8 to 10, F1037A; lane 11 to 13, R993A/F1037A) were incubated with 0.5 nM DNA substrate at 37 °C for 30 min. Reaction products were separated on 8% polyacrylamide gel. D indicates heat-denatured substrate control. (B) Quantitative analysis of (A). Experiments were repeated at least three times, error bars represent ± SD. (C) Electro mobility shift assay (EMSA) using forked duplex. 1, 2, 5 nM WRN wild type (lane 2 to 4) or WRN variants (lane 5 to 7, R993A; lane 8 to 10, F1037A; lane 11 to 13, R993A/F1037A) were incubated with 0.5 nM forked duplex substrate on ice for 30 min, then products were separated on 4 % polyacrylamide gel. (D) ATPase hydrolysis is disrupted by mutations. Representative polyethyleneimine thin-layer chromatography plate of ATP hydrolysis by wild type WRN or mutant proteins in the presence or in the absence of dsDNA is shown.