Salvage circulation of 5mC from calf DNA into new synthetic DNA

<p><strong>ABSTRACT</strong></p> <p>We investigated the salvage pathway of 5-methyl cytosine (5mC), a nucleotide modification crucial for epigenetic regulation, utilizing a mimicked natural pathway protocol. DNA hydrolysis revealed the presence of dCMP with 5mC modification (5mdCMP) in calf DNA, suggesting its potential recycling to the nucleotide pool. Phosphorylation experiments demonstrated the conversion of 5mdCMP to 5mdCTP, highlighting the feasibility of its salvage pathway. Nanopore sequencing confirmed the incorporation of salvaged 5mC into newly synthesized DNA strands. Additionally, protein expression assays revealed downregulation in the presence of salvaged 5mC, indicating its impact on gene expression. Our findings underscore the importance of the salvage pathway for 5mC and its implications for epigenetic regulation and disease. Further research is necessary to elucidate the mechanisms and consequences of 5mC salvage fully.</p>