Supplementary Figure 5: Loss of UV-DDB increases sensitivity to oxidant induced 8-oxoG, Related to Fig. 4.
From: Damage sensor role of UV-DDB during base excision repair
(a) Effect of UV-DDB on DNA ligase III activity in BER. A phosphorimage of the ligation step in BER is shown. The ligation reaction was performed in a 10 μl volume containing 50 mM HEPES, pH 7.5, 20 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 2 mM DTT, 2 mM ATP. The radiolabeled nicked DNA substrate (200 nM) was incubated with varying concentrations (0 to 400 nM) of DNA ligase III in the absence (-) or presence (+) of UV-DDB (50 nM). The incubation was at 37°C for 5 min. The reaction products were analyzed by denatured polyacrylamide gel electrophoresis. (b) Cell growth curves of normal and XPE lymphoblastoid cells treated with increasing concentrations of KBrO3. Data represents mean +/- SEM from three independent experiments, each performed in quadruplicates. (c) Western blot of BJ-hTERT cells transfected with scrambled or DDB2 siRNA and probed for DDB2, APE1 and OGG1, 72 hours post transfection. (d) Immunofluorescence images depicting mCherry-DDB2 recruitment to and departure from telomeres up to three hours after inducing damage. Graphs show DDB2 and OGG1 co-localization at telomeres. Scale bar: 5μm.
