Figure 3.

HNE adduction of WRN inhibits its helicase and exonuclease activities in vivo. Human normal (WT) or WS fibroblasts were pre-treated with HNE and the cell extracts were immunoprecipitated with rabbit anti-WRN antibody or control rabbit IgG. The immunoprecipitates were used in the measurements of WRN helicase (A), exonuclease (B and C) or ATPase activity (D) as described in Materials and Methods. Representative gels are shown. (E) Percent of helicase, exonuclease or ATPase activities from the data shown in A–D. Lanes 1, radiolabeled DNA substrate for helicase (A) or exonuclease (B and C) activity assays were reacted with control IgG immunocomplexes prepared from untreated WT fibroblasts. Lanes 2, the same as lanes 1, but cells were pre-treated with 100 μM HNE for 2 h. Lanes 3, the same as lanes 1, but substrates were reacted with immunocomplexes obtained against anti-WRN antibody. Lanes 4, the same as lanes 3, but cells were pre-treated with HNE. Lanes 5, the same as lane 4, but immunocomplexes were purified from WS fibroblasts. Lane 6, heat denatured helicase substrate. All data bars are the mean of three independent experiments with SDs indicated by error bars. Statistical analysis occurred via Student's t-test (***P < 0.001).