Single-Nucleotide Resolution Mapping of N6-Methyladenine in Genomic DNA

Abstract

N⁶-Methyladenine (6mA) is a naturally occurring DNA modification in both prokaryotes and eukaryotes. Herein, we developed a deaminase-mediated sequencing (DM-seq) method for genome-wide mapping of 6mA at single-nucleotide resolution. The method capitalizes on the selective deamination of adenine, but not 6mA, in DNA mediated by an evolved adenine deaminase, ABE8e. By employing this method, we achieved genome-wide mapping of 6mA in Escherichia coli and in mammalian mitochondrial DNA (mtDNA) at single-nucleotide resolution. We found that the 6mA sites are mainly located in the GATC motif in the E. coli genome. We also identified 17 6mA sites in mtDNA of HepG2 cells, where all of the 6mA sites are distributed in the heavy strand of mtDNA. We envision that DM-seq will be a valuable tool for uncovering new functions of 6mA in DNA and for exploring its potential roles in mitochondria-related human diseases.

Figure 1

Evaluation of the ABE8e-mediated deamination of dA and 6mA in DNA by ABE8e. (A) Workflow of the Endo V cleavage assay to assess the deamination of dA and 6mA. The dI formed from deamination of dA can be cleaved by Endo V. (B) Analysis of the 60-mer single A-DNA and single 6mA-DNA after ABE8e treatment at 37 °C for 30 min. The resulting DNA was incubated with Endo V at 37 °C for 1 h followed by polyacrylamide gel electrophoresis analysis. (C) LC-ESI-MS/MS analysis of dA, dI, and 6mA nucleosides from the 60-mer single A-DNA and single 6mA DNA with or without ABE8e treatment. (D) Steady-state kinetic parameters for deamination of dA or 6mA in DNA by ABE8e. (E) Michaelis–Menten plot showing the rate of ABE8e-mediated deamination versus the concentrations of the 60-mer single A-DNA. Data were fit with the Michaelis–Menten equation. (F) LC-ESI-MS/MS analysis of the remaining percentage of 6mA from different amounts of the 60-mer single 6mA DNA after ABE8e treatment, at 2.8 μM for 30 min.

Figure 2

Evaluation of the DM-seq method by sequencing. (A) Schematic illustration of deaminase-mediated mapping of 6mA in DNA. (B) Sanger sequencing of 314-bp A-DNA and 314-bp 6mA-DNA with or without ABE8e treatment. (C) Quantitative measurements of A-to-G conversion rates and evaluation of the 6mA readout in the 314-bp 6mA-DNA cloned after ABE8e treatment followed by colony sequencing. Fifty-three colonies were picked for sequencing. (Figure S8). (D) Statistical analysis of the deamination rate of dA with different flanking nucleobases of 314-bp 6mA-DNA by colony sequencing.

Figure 3

Genome-wide mapping of 6mA in E. coli by DM-seq. (A) LC-ESI-MS/MS analysis of the remaining percentages of dA and 6mA in genomic DNA of wild-type and dam-deficient E. coli strains after ABE8e treatment. Each percentage is relative to the amount in the respective strains prior to ABE8e treatment. (B) Motif sequence profile and sequence conservation analysis. (C) Heat map showing the correlation of the identified 6mA sites in different replicates. (D) Numbers of 6mA sites identified in the genomes (from both DNA strands) of wild-type and dam-deficient E. coli strains. (E) Theoretical numbers of GATC motifs (gray column) and numbers of identified G6mATC motifs in the genomes of wild-type (red column) and dam-deficient (blue column) E. coli strains. G6mATC from duplex DNA was recognized as one site. (F) Representative view of 6mA sites in the genome of the wild-type E. coli strain (position: 98809 to 98829). Red asterisks denote 6mA sites. Red and blue columns represent the number of reads with A and G at the indicated sites, respectively. (G) Sanger sequencing of three different 6mA sites with or without ABE8e treatment in the genomes of wild-type and dam-deficient E. coli strains.

Figure 4

Genome-wide mapping of 6mA in mtDNA by DM-seq. (A) LC-ESI-MS/MS for evaluating the levels of dA and 6mA in human HepG2 mtDNA after ABE8e treatment. ABE8e treatment led to the production of dI from dA. (B) Evaluation of the enrichment fold for mtDNA by qPCR. (C) Circos plot showing the distribution of 6mA sites across human HepG2 mtDNA. The inner three circles represent three biological replicates; the outer circle represents 6mA sites (red bars). (D) Representative view of 6mA sites in human HepG2 mtDNA (positions 5443 to 5454). Red asterisks denote 6mA sites. Red and blue columns represent the respective numbers of A and G reads at the indicated positions.