Biochemical analysis of activation-induced cytidine deaminase (AID) from bony fish: insights into the mechanisms of AID

Abstract

The properties of Activation-induced cytidine deaminase (AID) necessary for its role in immunity and lymphoma generation are currently unknown. AID mutants and species-variants, as compared to wild-type human AID, were identified so as to map out responsible residues/domains. Variant AIDs were analyzed by alkaline cleavage and EMSA. AID from bony fish species (Ictalurus punctatus and Danio rerio) were identified as the most divergent from human AID. Dr-AID acted enzymatically similar to H-AID, while Ip-AID was less mutagenic. Point mutations within Ip AID were generated, substituting the human counterpart amino acid. One mutant rescued Ip-AID enzyme activity to levels seen in H-AID and Dr-AID. The reverse mutation in H-AID almost fully abolished the deaminating ability of H-AID. DNA-binding kinetics were tested at different temperatures to analyze if the disparity in activity levels was due to binding of DNA. The single mutation in IP-AID increases the affinity of AID to bind ssDNA and this effect is substantially more pronounced at lower temperatures. These results have provided novel information on AID domains/residues necessary for its function.