Fig. 1. Localisation of pol following DNA damage. (A) Foci formation frequencies of pol after DNA damaging (UV irradiation, BaP and -irradiation) or replication inhibitory (HU) treatments. MRC5 cells from our laboratory (GDSC, open bars) or from the laboratory of Bergoglio and colleagues (Tou, shaded bars) (Bergoglio et al., 2002) were transfected with eGFPpol constructed in our laboratory (GFPK, black), eGFP-C2-HsPOLK constructed in the laboratory of Bergoglio (GFPK-Tou, blue), or eGFPpol (GFPH, red) and incubated for 20 hours. Cells were then treated with the indicated doses of damaging agents and further incubated for the indicated times. The proportion of eGFPpol (or eGFPpol)-expressing cells in which the protein was localised in nuclear foci was determined. All experiments were carried out in triplicate and each data point is the mean of three independent scorings. Benzo[a]pyrene (BaP) and -ray experiments were carried out only with cells and plasmid from our laboratory. Error bars indicate standard error. (B,C) Typical images of cells expressing eGFPpol (B), or eGFPpol (C) 6 hours after 10 J/m² UV irradiation. (D) Sub-nuclear pol protein localisation after UV irradiation. MRC5 cells were transfected with pEGFPpol, or pEGFPpol and incubated for 20 hours. Cells were then UV irradiated with 10 J/m² and incubated for 6 hours. Cellular proteins were fractionated into detergent extractable (unbound, UB), salt extractable nuclear matrix binding fraction (NcB) and a fraction resistant to salt extraction (chromatin binding, ChrB) and analysed by immunoblotting.