Single-Nucleotide Resolution Analysis of 5-Hydroxymethylcytosine in DNA by Enzyme-Mediated Deamination in Combination with Sequencing.

The report of the existence of 5-hydroxymethylcytosine (hmC) in mammalian genomes is a milestone discovery. hmC is now generally viewed as the sixth base of DNA with important functions on epigenetic regulation. The in-depth investigation of the biological functions of hmC requires elucidating the distribution patterns of hmC in genomes, better in single-nucleotide resolution. It was reported that the cytosine deaminases of the APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide-like) family are nucleic acid editing enzymes and can deaminate cytosine (C) to form uracil (U). Particularly, a subfamily of APOBEC (APOBEC3A) can efficiently deaminate both C and 5-methylcytosine (mC). In the current study, we identified that APOBEC3A protein can effectively deaminate C, mC, and hmC but shows no observable deamination activity toward glycosylated hmC (β-glucosyl-5-hydroxymethyl-2'-deoxycytidine, ghmC) by using the restriction enzyme-based assay and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. By virtue of the differential deamination activity of APOBEC3A toward C, mC, and ghmC in conjugation with sequencing, we developed the single-nucleotide resolution analysis of hmC in DNA. In this analytical strategy, the original C and mC in DNA will be deaminated by APOBEC3A to form U and thymine (T), both of which will read as T during sequencing, while ghmC is resistant to deamination and will read as C during sequencing. Therefore, the remaining C in the sequence context only could come from original hmC, which offers the single-nucleotide resolution analysis of hmC in DNA. This APOBEC3A-mediated deamination sequencing (AMD-seq) is straightforward and involves no bisulfite treatment, which avoids the substantial degradation of DNA. Future application of this strategy can be performed for the reliable mapping of hmC in genome-wide scale at the single-nucleotide resolution.