In vitro deamination assay to measure the activity and processivity of AID/APOBEC enzymes

The AID/APOBEC family of enzymes are cytidine/deoxycytidine deaminases that primarily catalyze the deamination of deoxycytidines (dCs) into deoxyuridines (dUs) on single-stranded DNA (ssDNA). In humans, there are 11 members within the family. AID and APOBEC3 (A3) enzymes have been extensively characterized for their ability to introduce promutagenic dUs during antibody gene diversification and intrinsic immune defenses against viruses and retrotransposons, respectively. In order to search for a local dC deamination target to effectively catalyze the deamination reaction, AID/APOBEC enzymes adopt facilitated diffusion as a mechanism to search for the target deamination sites on ssDNA substrates, which includes one-dimensional (1D) movements termed sliding, and three-dimensional (3D) movements termed jumping and intersegment transfer. This type of diffusional mechanism enables AID/APOBEC enzymes to processively scan ssDNA substrates and serves as a key determinant to the mutagenic potential of AID/APOBEC enzymes in vivo. The catalysis and processive ssDNA scanning behaviors of AID/APOBEC enzymes can be assessed using purified proteins and synthetic ssDNA through an in vitro deamination assay. In this Chapter, we describe how to perform deamination assays where DNA scanning mechanisms and processivity can be measured under single-hit conditions using a fluorescently labeled ssDNA substrate. The in vitro deamination assay can also be applied to determine AID/APOBEC activity in cell lysates or in kinetic reactions to determine the specific activity of purified enzymes.