FIGURE 2.

Analysis of deamination activity of WT and mutant AID on an ssDNA oligonucleotide substrate. A, schematic representation of the C-deamination assay. A ³²P-labeled 36-nt substrate was incubated with AID followed by UDG and hot alkali treatment to generate a 14-nt fragment product. B, screening for deamination activity for AID HIGM-2 missense mutants. WT AID (10 nm) or mutant AID (10 to 100 nm) was incubated with ssDNA substrate for 60 min at 37 °C. The appearance of a 14-nt fragment product demonstrates that three AID missense mutants (S43P, L98R, and R174S) retain deamination activity with specific activities shown in C. D and E, analysis of ssDNA deamination (D) and specific activity (E) of C-terminal deletion AID mutants. AID protein concentrations and incubation times used to calculate specific activities are described under “Experimental Procedures.” Error bars represent ±1 S.E. derived from ≥3 independent measurements.