Figure 4.

BsuLigD is endowed with an AP lyase activity. (A) Analysis of the capacity of BsuLigD to incise an internal natural abasic site. The [α³²P]3′-labeled 2′-deoxyuridine-containing substrate was treated with 27 nM E. coli UDG (lane c), leaving an intact AP site. The resulting AP-containing DNA was incubated in the presence of either 5 nM hAPE1 that cleaves 5′ to the AP site, 3.5 nM EndoIII that incises 3′ to the AP site, or increasing concentrations of BsuLigD (0, 29, 57 and 114 nM) for 1 h at 30°C, as described in Materials and Methods. After incubation samples were analyzed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. (B) Analysis of the capacity of BsuLigD to incise an internal tetrahydrofuran (H). The 3′ [α³²P]3′-dAMP labeled oligonucleotide containing the lyase-resistant analogue tetrahydrofuran (H) was incubated in the presence of either hAPE1, EndoIII or increasing concentrations of BsuLigD as described above. Position corresponding to the products 16-mer 5′-dRP and 16-mer 5′-P is indicated. The figure is a composite image made from different parts of the same experiment.