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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. 

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.