Pmt1-dependent and state-restricted DNA methylation in fission yeast

DNA 5-methylcytosine (5mC) is widely considered absent from budding and fission yeasts. The Dnmt2 family enzyme Pmt1 in Schizosaccharomyces pombe is annotated as a tRNA C38 methyltransferase, but whether it methylates DNA in vivo has remained unresolved. Using orthogonal chemistry (HPLC/LC-MS and 1H-NMR) with external standards (limit of detection for 5mdC = 0.0125 mM), we detect authentic 5-methyl-2’- deoxycytidine (5mdC) in enzymatically digested genomic DNA from S. pombe . Quantification across isogenic strains shows that 5mdC is undetectable in vegetative wild type but rises to 0.518% (plus or minus 0.025) of cytosines two hours after G0 exit. Loss of the cytosine/5-mC deaminase Fcy1 causes marked accumulation: 1.445% (plus or minus 0.361) in vegetative cells and 5.943% (plus or minus 1.364) at 2 hours, approximately 11.5-fold over wild type. By contrast, pmt1Δ and fcy1Δ pmt1Δ remain below detection in all conditions, establishing Pmt1 as the DNA-directed methyltransferase in vivo. Nascent-strand fractionation and lambda exonuclease enrichment place this transient 5mdC pulse on Okazaki-enriched DNA, consistent with co- replicative installation. Functionally, the first S phase after quiescence shows increased Rad22-YFP foci in fcy1Δ , which are dampened by queuine ; in a sensitized background ( ung1Δ thp1Δ ), deleting fcy1 reduces C->T transitions by about 30 percent. Together, chemical, genetic, and temporal evidence reveals a Pmt1- dependent, state-restricted DNA methylation program in fission yeast that is rapidly curtailed by Fcy1, redefining the epigenetic landscape of S. pombe and providing a minimal, tractable system to dissect regulated DNA methylation in eukaryotes.