AID/APOBEC family cytosine deaminases canonically play crucial roles in immunity by converting cytosine to uracil in single-stranded DNA (ssDNA) in specific sequence contexts. Outside of this established physiological role, AID/APOBEC enzymes have also been implicated in the poorly-understood process of DNA demethylation through their proposed deamination of epigenetically-modified cytosine bases like 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). However, there has been no thorough biochemical characterization of AID/APOBEC activity on these substrates, or on the recently-discovered 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) to inform this proposed role. Here, we provide the first steady-state kinetic measurements of the most active family member–APOBEC3A (A3A)–against various natural and unnatural modified substrates using a novel, restriction-enzyme-based deaminase assay. In this analysis, we found that A3A has an enhanced activity against 5mC that distinguishes it from the other AID/APOBEC family members, which discriminate against substrates via a purely steric mechanism. We also concluded that A3A has poor activity against larger oxidized cytosines, like 5hmC, 5fC, and 5caC, and therefore likely does not contribute substantially to active DNA demethylation via deamination of these bases. Importantly, given our discovery of A3A’s vast preference for unmodified C and 5mC over larger bases like 5hmC, 5fC, and 5caC, we hypothesized that A3A could be utilized in an enzymatic, nondestructive alternative to bisulfite sequencing-based methods (APOBEC-coupled Epigenetic Sequencing, or ACE-Seq) to localize genomic 5mC or 5hmC at single-base resolution with up to 1000-fold less starting material. Though much optimization remains, initial validation of ACE-Seq on genomic DNA from mutant T4 phage has fueled the plausibility of this method. By localizing epigenetic bases in important yet unexplored cell populations, this research will help refine the currently poorly-understood process of active DNA demethylation and our broader paradigms of genetic regulation.

Defining and exploiting APOBEC3A's cytidine deaminase activity on the extended epigenome

251st National Meeting (2016)

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