Fig. 4

Comparison of the catalytic rate of Atlantic cod AID with other AID orthologs. A The catalytic rate of Gm-AID was compared to that of other AID orthologs through Michaelis–Menten kinetics determined by the alkaline cleavage assay. Three independent protein preparations of each AID ortholog were tested in duplicate. Data is represented as mean ± SEM (n = 6). B The relative catalytic activity of Gm-AID was confirmed through a PCR-based deamination assay using a single-stranded plasmid as the substrate. The presence of a PCR amplicon indicates AID-mediated deamination events. Through serial dilution analysis of the substrate incubated with AID which is used as the template for the deamination-specific PCR, it was apparent that Dr-AID is 10–100-fold more active than Hs-AID, while Gm-AID supported 100- and 10,000-fold less mutation levels than Hs-AID and Dr-AID, respectively. Four independent experiments were performed, and the presence of a PCR band in each experiment was recorded as a black dot below each lane in the representative gel