Fig. 2. (A) Expression and purification of the recombinant His-tagged Pa-UDGb (see Materials and methods). I, total extract of the E.coli strain BL21(DE3)pET28c(+)-paudgb; II, total extract of the same cells, following induction with IPTG; III, cleared lysate of the same cells; IV, proteins eluted from the Ni-NTA column with 250 mM imidazole; V, Pa-UDGb eluted from a Mono-S column; M, molecular size marker. The panel shows a 12% Coomassie blue-stained denaturing polyacrylamide gel. (B) Processing of G·U mispairs by fraction IV obtained from E.coli BL21 cells transfected with the pET28c(+)-paudgb plasmid (lane 2) or with the empty pET28c(+) vector (lane 3). This experiment shows that no E.coli uracil-processing activity is present in this fraction. The 60mer oligonucleotide substrate G·U was incubated for 1 h at 37°C with 6 µl of fraction IV as described in Materials and methods. (C) Pa-UDGb is a heat-stable, monofunctional uracil-DNA glycosylase. The enzyme alone removes uracil at both indicated temperatures, but does not cleave the sugar–phosphate backbone of the mispaired DNA substrate, as witnessed by the absence of the 23mer product band in the reaction where the G·U substrate was treated with Pa-UDGb alone (lane 1). Cleavage occured only upon the addition of hot alkali (lanes 2 and 6) or of human HAP1 (lane 3). The faint product band in lane 5 is due to heat-induced spontaneous β-elimination at the labile AP sites. Incubation at 70°C significantly increased the activity of Pa-UDGb (lane 6), whereas the E.coli UDG was completely inactivated at this temperature (lane 7, cf. lane 4).