Figure 7.

Rad18-Ub fusions are unable to respond to DNA damage. (A) Knockdown of SHPRH and overexpression of Rad18-Ub is epistatic. SupF reporter plasmid (0.5% MMS) was recovered from HEK 293T cells 48 h after cotransfection with the indicated GFP-Rad18 constructs (Ub, ubiquitin) and siRNAs (siCtrl, control siRNA). Data represent mean and standard deviations from three independent experiments. At least 2,000 colonies were analyzed per condition. **, P < 0.01, relative to control knockdown. (B) Rad18-Ub chimeras are nuclear but fail to localize to sites of DNA damage. U2OS clones stably expressing different forms of GFP-Rad18 were treated with 0.005% MMS for 4 h before preextraction and fixation. Rad18 was visualized using direct GFP fluorescence, γ-H2AX was detected using immunofluorescence. Bars, 10 µm. (C) Rad18-Ub chimeras do not form foci after any type of DNA damage. Clones in B were treated with MMS for 4 h or allowed to recover for 4 h after exposure to the indicated doses of UV or ionizing radiation (IR). GFP-Rad18 foci were counted using ImageJ. Each bar represents a mean and standard deviation from three independent experiments. At least 100 cells were counted per condition. **, P < 0.01, relative to wild-type (WT) Rad18. (D) Rad18-Ub chimeras cannot induce PCNA ubiquitination. Rad18^−/− HCT116 cells were transiently transfected with GFP-Rad18 chimera constructs and damaged with UV or MMS before being analyzed as in Fig. 2 C. (E) Rad18-Ub chimeras cannot suppress mutagenesis on MMS- or UV-damaged plasmids. SupF reporter plasmid (0.5% MMS or 1,000 J/m² UV) was recovered from Rad18^−/− HCT116 cells 48 h after cotransfection with the indicated GFP-Rad18 chimera constructs (Ub(I44A), ubiquitin-I44A). Data represent means and SEMs from four independent experiments. At least 2,000 colonies were analyzed per condition. *, P < 0.05, relative to GFP-only control. R18, Rad18.