Biology subjects

MILED, S.

Publications and source records attributed to MILED, S..

3 recordsLinked to original sources

bioRxiv2025

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, pmt1{Delta} and fcy1{Delta} pmt1{Delta} 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 fcy1{Delta}, which are dampened by queuine ; in a sensitized background (ung1{Delta} thp1{Delta}), 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.

molecular biology↗

bioRxiv2025

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

BackgroundThe 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. ResultsWe 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 (Dnmt2Dm) or Plasmodium falciparum TRDMT1 (TRDMT1Pf) 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. ConclusionAcross 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.

molecular biology↗

bioRxiv2025

"Genome Stability under Silence: DNA Repair Networks in Quiescent Fission Yeast"

Most of our current understanding of genome integrity derives from studies in proliferating cells, yet most somatic cells in multicellular organisms reside in non-dividing, quiescent states. Using Schizosaccharomyces pombe, we dissected the mechanisms by which quiescent cells maintain genome stability in the absence of DNA replication. Combining time-resolved mutational analyses, DNA damage assays, and genetic dissection of DNA repair pathways, we found that quiescent cells progressively accumulate distinct types of spontaneous lesions-particularly uracil residues, abasic sites, and ribonucleotide insertions-which are counteracted by a modular network of repair mechanisms. Base excision repair (BER), ribonucleotide excision repair (RER), and R-loop resolution each contribute uniquely to genome surveillance in G0. We show that uracil incorporation becomes a predominant threat under quiescent conditions, especially when nucleotide pools are imbalanced. BER-deficient mutants (e.g., nth1{Delta}, ung1{Delta}) exhibit mutation spectra dominated by C: G > T: A transitions and oxidative lesions, while synthetic combinations reveal compensatory or epistatic interactions. Using single-cell micromanipulation and viability assays, we show that specific gene deletions (e.g., hnt3{Delta}rhp52{Delta}, sen1{Delta}rad13{Delta}) severely compromise post-quiescence recovery, underscoring the importance of cooperative DNA repair even in non-replicative contexts. Our results delineate a functionally compartmentalized hierarchy of DNA repair activities during quiescence, providing a new framework to understand how non-dividing cells limit genome instability, with implications for aging, cancer dormancy, and neurodegeneration.

cell biology↗