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Abstract N6‐adenine (6mA) DNA methylation plays an important role in gene regulation and genome stability. The 6mA methylation inTetrahymena thermophilais mainly mediated by the AMT complex, comprised of the AMT1, AMT7, AMTP1, and AMTP2 subunits. To date, how this complex assembles on the DNA substrate remains elusive. Here we report the structure of the AMT complex bound to the OCR protein from bacteriophage T7, mimicking the AMT–DNA encounter complex. The AMT1–AMT7 heterodimer approaches OCR from one side, while the AMTP1 N‐terminal domain, assuming a homeodomain fold, binds to OCR from the other side, resulting in a saddle‐shaped architecture reminiscent of what was observed for prokaryotic 6mA writers. Mutation of the AMT1, AMT7, and AMTP1 residues on the OCR‐contact points led to impaired DNA methylation activity to various extents, supporting a role for these residues in DNA binding. Furthermore, structural comparison of the AMT1–AMT7 subunits with the evolutionarily related METTL3–METTL14 and AMT1–AMT6 complexes reveals sequence conservation and divergence in the region corresponding to the OCR‐binding site, shedding light on the substrate binding of the latter two complexes. Together, this study supports a model in which the AMT complex undergoes a substrate binding‐induced open‐to‐closed conformational transition, with implications in its substrate binding and processive 6mA methylation. 

Abstract N⁶-adenine methylation occurs in both DNA and RNA (referred to as 6mA and m6A, respectively). As an extensively characterized epi-transcriptomic mark found in virtually all eukaryotes, m6A in mRNA is deposited by METTL3-METTL14 complex. As a transcription-associated epigenetic mark abundantly present in many unicellular eukaryotes, 6mA is coordinately maintained by two AMT1 complexes, distinguished by their mutually exclusive subunits, AMT6 and AMT7. These are all members of MT-A70 family methyltransferases (MTases). Despite their functional importance, no structure for holo-complexes with cognate DNA/RNA substrate has been resolved. Here, we employ AlphaFold3 (AF3) and molecular dynamics (MD) simulations for structural modeling ofTetrahymenaAMT1 complexes, with emphasis on ternary holo-complexes with double-stranded DNA (dsDNA) substrate and cofactor. Key structural features observed in these models are validated by mutagenesis and various other biophysical and biochemical approaches. Our analysis reveals the structural basis for DNA substrate recognition, base flipping, and catalysis in the prototypical eukaryotic DNA 6mA-MTase. It also allows us to delineate the reaction pathway for processive DNA methylation involving translocation of the closed form AMT1 complex along dsDNA. As the active site is highly conserved across MT-A70 family of eukaryotic 6mA/m6A-MTases, the structural insight will facilitate rational design of small molecule inhibitors, especially for METTL3-METTL14, a promising target in cancer therapeutics. 

Abstract DNA modifications, such as N6-methyladenine (6mA), play important roles in various processes in eukaryotes. Single-molecule, real-time (SMRT) sequencing enables the direct detection of DNA modifications without requiring special sample preparation. However, most SMRT-based studies of 6mA rely on ensemble-level consensus by combining multiple reads covering the same genomic position, which misses the single-molecule heterogeneity. While recent methods have aimed at single-molecule level detection of 6mA, limitations in sequencing platforms, resolution, accuracy, and usability restrict their application in comprehensive epigenetic studies. Here, we present SMAC (single-molecule 6mA analysis of CCS reads), a novel framework for accurately detecting 6mA at the single-molecule level using SMRT circular consensus sequencing (CCS) data from the Sequel II system. It is an automated method that streamlines the entire workflow by packaging both existing softwares and built-in scripts, with user-defined parameters to allow easy adaptation for various studies. By utilizing the statistical distribution characteristics of enzyme kinetic indicators on single DNA molecules rather than a fixed cutoff, SMAC significantly improves 6mA detection accuracy at the single-nucleotide and single-molecule levels. It simplifies analysis by providing comprehensive information, including quality control, statistical analysis, and site visualization, directly from raw sequencing data. SMAC is a powerful new tool that enables de novo detection of 6mA and empowers investigation of its functions in modulating physiological processes. 

Enabled by long-read sequencing technologies, particularly Single Molecule, Real-Time sequencing, N6-methyladenine (6mA) footprinting is a transformative methodology for revealing the heterogenous and dynamic distribution of nucleosomes and other DNA-binding proteins. Here, we present ipdTrimming, a novel 6mA-calling pipeline that outperforms existing tools in both computational efficiency and accuracy. Utilizing this optimized experimental and computational framework, we are able to map nucleosome positioning and transcription factor occupancy in nuclear DNA and establish high-resolution, long-range binding events in mitochondrial DNA. Our study highlights the potential of 6mA footprinting to capture coordinated nucleoprotein binding and to unravel epigenetic heterogeneity. 

Stable inheritance of DNA N⁶-methyladenine (6mA) is crucial for its biological functions in eukaryotes. Here, we identify two distinct methyltransferase (MTase) complexes, both sharing the catalytic subunit AMT1, but featuring AMT6 and AMT7 as their unique components, respectively. While the two complexes are jointly responsible for 6mA maintenance methylation, they exhibit distinct enzymology, DNA/chromatin affinity, genomic distribution, and knockout phenotypes. AMT7 complex, featuring high MTase activity and processivity, is connected to transcription-associated epigenetic marks, including H2A.Z and H3K4me3, and is required for the bulk of maintenance methylation. In contrast, AMT6 complex, with reduced activity and processivity, is recruited by PCNA to initiate maintenance methylation immediately after DNA replication. These two complexes coordinate in maintenance methylation. By integrating signals from both replication and transcription, this mechanism ensures the faithful and efficient transmission of 6mA as an epigenetic mark in eukaryotes. 

Although DNAN⁶-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 6mA transmission mechanism remains to be elucidated. Taking advantage of single-molecule real-time circular consensus sequencing (SMRT CCS), here we provide definitive evidence for semiconservative transmission of 6mA inTetrahymena thermophila. In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2′-deoxyuridine (BrdU). In ΔAMT1cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, whereas de novo methylation in ΔAMT1cells is slow and sporadic. InTetrahymena, regularly spaced 6mA clusters coincide with the linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by the reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semiconservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a 6mA transmission pathway with a striking similarity to 5-methylcytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.