Fig. 3.

DNA strand breaks, UNG substrates and NER. (A) Comet assays: HeLa cells were treated with 100 μM SIdU (left panel) or SBrdU (right panel) for 48 h and UVA irradiated. DNA strand breaks and/or abasic sites were determined by single cell electrophoresis with or without digestion with recombinant UNG as indicated. (B) Sensitivity of ung^−/− MEFs: UNG-proficient (WT) and -deficient (UNG) cells were grown in the presence of SBrdU (5 and 30 μM, respectively; left panel) or SIdU (10 and 60 μM; right panel) for 24 h to ensure equal levels of DNA substitution (0.025% for SBrdU,0.022% for SIdU). They were UVA irradiated and survival was determined 7 days later by MTT assay. (C) 6:4 Py:Py excision: HeLa cells grown in 200 μM SIdU or 300 μM SBrdU for 48 h were irradiated as indicated with 20 kJ/m² UVA followed by 20 J/m² UVC and returned to normal growth medium. DNA was extracted at the times indicated and 6-4 Py:Py photoproducts were quantified by ELISA. (D) Sensitivity of NER-defective cells: MRC5VA (NER-competent), XP12RO (XPA, NER-deficient) and XP129 (NER proficient XP ‘revertant’) cells were incubated with 120 μM SBrdU (left panel) or SIdU (right panel) for 72 h and UVA irradiated. Survival was measured by MTT assay 10 days later. In the absence of drug treatment, survival of all three cell lines was >90% at all UVA doses used. In the experiment shown (representative of two), DNA substitution by the three cell lines was similar at 0.035% of TdR for SBrdU and 0.02% for SIdU.