Figure 2. Pol λ ^−/−/pol β ^−/− double knock-out cell extracts show strongly reduced in vitro BER activity.

Experiments were conducted as described under Materials and Methods. In vitro BER time course experiments showing the capacity of wild-type (lanes 1–3), pol λ −/− (lanes 4–6), pol β −/− (lanes 7–9) and pol λ ^−/−/pol β ^−/− (lanes 10–12) MEF cell extracts to repair a solitary base lesion in double-stranded DNA, the uracil (U) lesion opposite G (A) or the 8-oxoguanine (8-oxodG lesion opposite C (B). The schematic at the top in each panel illustrates the DNA substrate, and the reaction mixtures contained [α-³²P]dCTP and [α-³²P]dGTP, respectively. The BER reaction mixtures were incubated for 5 (lanes 1, 4, 7 and 10), 10 (lanes 2, 5, 8, and 11) or 30 (lanes 3, 6, 9 and 12) min, respectively. Photographs of autoradiograms after denaturing PAGE are shown illustrating incorporation of [³²P]dCMP (A) or [³²P]dGMP (B) into the fully repaired DNA. The positions of the fully repaired and ligated BER products are indicated. (C) Quantification of the in vitro BER activity of the cell extracts used in panels A and B. Average values are shown for the respective extracts.