Cross-species DNMT2-mediated DNA methylation with an S-phase 5mC pulse

Background

The budding yeast Saccharomyces cerevisiae lacks endogenous DNA methyltransferases, providing a DNMT-free chassis to test catalytic potential of candidate enzymes. Building on our discovery that the Schizosaccharomyces pombe Dnmt2 ortholog Pmt1 can methylate DNA in vivo, we asked whether Dnmt2-family enzymes install genomic 5-methyl-2′-deoxycytidine (5mdC) across heterologous contexts.

Results

We fused Pmt1 to an N-terminal SNAP tag and expressed it in S. cerevisiae , where it was well expressed, nuclear-enriched, and non-toxic. LC/MS of genomic nucleosides revealed readily detectable 5mdC in Pmt1–SNAP cells but not detected in empty-vector controls. Following G1 arrest–release, 5mdC rose at S phase and, unlike in S. pombe , failed to decay over the assayed window, indicating limited methyl-cytosine clearance in the DNMT-free chassis. Extending this paradigm, expression of Drosophila melanogaster Dnmt2 (Dnmt2 ^Dm ) or Plasmodium falciparum TRDMT1 (TRDMT1 ^Pf ) in S. cerevisiae similarly yielded genomic 5mdC. In vivo, Drosophila manipulations supported a DNA-directed function for Dnmt2, and in vitro S2-cell assays using two independent synchronizations showed coordinated Dnmt2 transcript induction (RT-qPCR) with an accompanying methyl-DNA signal.

Conclusion

Across yeast, fly, and parasite orthologs, Dnmt2-family enzymes act as bona fide DNA cytosine methyltransferases. The S. cerevisiae chassis reveals an S-phase–linked 5mdC pulse that persists in the absence of native turnover pathways, offering a minimal, genetically tractable system to dissect substrate selection, cell-state gating, and methyl-cytosine clearance for this atypical DNMT family.

Extending this paradigm, expression of Drosophila Dnmt2 or Plasmodium falciparum TRDMT1 in S. cerevisiae similarly yielded genomic 5mdC, consistent with the first in vivo report that TRDMT1 can methylate DNA in the parasite.