Figure 5.

Depletion of Top1 protects human cells from alkylation-induced DNA damage. (a) Control ‘WT’, TDP1^KD, Top1^KD or TDP1^KD/Top1^KD double mutant cells in which Top1 level was additionally depleted by siRNA were incubated with increasing concentrations of MMS for 15 min at 37°C. Survival was determined from three biological replicates and presented as mean ± s.e.m. Top1 depletion protects TDP1^KD from MMS damage (Asterisks; P < 0.05; t-test between TDP1^KD ‘closed circles’ and TDP1^KD Top1^KD cells ‘closed squares’). This protection did not fully restore resistance to levels observed in Top1^KD cells (Stars; P < 0.01; t-test between Top1^KD ‘open squares’ and TDP1^KD Top1^KD cells ‘closed squares’), suggesting that a proportion of MMS-induced breaks are processed by TDP1 in a Top1-independent manner. (b) Human MRC5 cells were incubated with 20 μM CPT or with 2 μg/ml MMS for 3 h at 37°C and cell lysate fractionated by SDS–PAGE and analyzed by immunoblotting. (c) MRC5 cells were incubated with DMSO or 50 μM 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole ‘DRB’ for 1 h or with 1 μg/ml α-amanitin for 16 h followed by an additional 3 h incubation with 2 μg/ml MMS. Cell lysates were fractionated by SDS–PAGE and analyzed by immunoblotting.