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AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation

Abstract

Activation-induced deaminase (AID)/APOBEC–family cytosine deaminases, known to function in diverse cellular processes from antibody diversification to mRNA editing, have also been implicated in DNA demethylation, a major process for transcriptional activation. Although oxidation-dependent pathways for demethylation have been described, pathways involving deamination of either 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) have emerged as alternatives. Here we address the biochemical plausibility of deamination-coupled demethylation. We found that purified AID/APOBECs have substantially reduced activity on 5mC relative to cytosine, their canonical substrate, and no detectable deamination of 5hmC. This finding was explained by the reactivity of a series of modified substrates, where steric bulk was increasingly detrimental to deamination. Further, upon AID/APOBEC overexpression, the deamination product of 5hmC was undetectable in genomic DNA, whereas oxidation intermediates remained detectable. Our results indicate that the steric requirements for cytosine deamination are one intrinsic barrier to the proposed function of deaminases in DNA demethylation.

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Figure 1: Proposed noncanonical role for AID/APOBEC enzymes acting on modified cytosine substrates in DNA.
Figure 2: AID/APOBEC enzymes preferentially deaminate unmodified cytosine.
Figure 3: DNA deamination decreases as a function of increasing steric bulk at the 5 position of cytosine.
Figure 4: AID/APOBEC enzymes do not perturb levels of 5mC oxidation intermediates in genomic DNA.

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Acknowledgements

We are grateful to M. Bartolomei, M. Weitzman and M. Lazar for helpful discussions, K. Gajula and S. Manning for technical assistance and the University of North Carolina Biomarker Mass Spectrometry Facility for guidance. We are also grateful for A. Drohat (University of Maryland), A. Guminski (Johns Hopkins University) and J. Guo and H. Song (Johns Hopkins University) for providing reagents. This work was supported in part by the Rita Allen Foundation (to R.M.K.), the W. W. Smith Charitable Trust (to R.M.K.) and US National Institutes of Health grants K08-AI089242 (to R.M.K.), GM056834 (to J.T.S.) and U01DK089565 (to Y.Z.). Y.Z. is an investigator of the Howard Hughes Medical Institute.

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R.M.K., J.T.S. and Y.Z. conceived the project. R.M.K., J.T.S., Y.Y., C.S.N. and Y.Z. designed the experiments. C.S.N., H.J., Y.Y., L.S. and H.L.G. performed the experiments. C.S.N., H.J. and L.S. analyzed the data. C.S.N., H.J., L.S., H.L.G., J.T.S., Y.Z. and R.M.K. interpreted the data. C.S.N., J.T.S. and R.M.K. wrote the manuscript, and all authors edited the manuscript.

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Correspondence to Yi Zhang or Rahul M Kohli.

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Nabel, C., Jia, H., Ye, Y. et al. AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation. Nat Chem Biol 8, 751–758 (2012). https://doi.org/10.1038/nchembio.1042

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