Figure 4.

N-glycosylase activity assays of AlkA and Endo VIII for Xan. (A) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. (B) HPLC analysis of [³H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [³H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released ³H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions containing the released ³H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). (C) HPLC analysis of [³H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.