Figure 4.

Cells downregulating polι show an impaired uracil-initiated base excision DNA repair synthesis in human cell-free extracts. (A) MRC5 cells were transfected with scrambled siRNAs (lane 1) or polι-specific siRNAs (lanes 2 and 4) or with polβ-specific siRNAs (lanes 3 and 4). After 72 h, cells were harvested and extracts were separated by SDS–PAGE. Immunoblots were incubated with anti-polι or anti-polβ antibodies. β-Actin detection was used as a loading control. (B) MRC5 cells were transfected with a pool of polι-specific siRNAs 24 h before treatment with increasing doses of H₂O₂ for 10 min at 4°C. Cells were counted 72 h later. Percentage of control growth was plotted for each data point, representing the mean of three independent experiments. (C) Denaturing gel of G·U repair products after incubation with siRNA-transfected MRC5 whole-cell extracts: 50 nM of G·U was incubated with 10 μg extract at 37°C, and aliquots were withdrawn at different time intervals and analysed on a 20% denaturing PAGE. Formation of 40 mer repair product was quantified by phosphorimaging. (D) The average initial rate of BER DNA repair synthesis in whole-cell extracts. For comparison, the DNA repair synthesis in extract from cells transfected with scrambled siRNA (NT) was assumed as 100%. Error bars show the s.d. of the mean of three independent experiments.