5-Methylcytosine (mC) is a stable epigenetic modification of DNA that plays a key role in controlling gene expression. Symmetric methylation of cytosines within CpG dinucleotides permits the inheritance of methylation patterns through DNA replication. Maintenance DNA methyltransferase (DNMT1) acts at hemimethylated CpGs created by semiconservative replication, maintaining tissue specific DNA methylation patterns. The removal of DNA methylation marks is mediated by ten eleven translocation (Tet) dioxygenases which iteratively oxidize the methyl group to give 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC). Although it is known that DNMT1 activity is affected by mC oxidation, the kinetics of DNA methylation in the presence of these oxidized forms of mC had not been determined, and the structural basis of the diminished methyltransferase activity is unknown.<br/>In the present work, we investigated the kinetics of DNMT1-mediated methylation in the presence of oxidized mC, hmC, fC, caC, and related mC analogues with extended alkyl chains. Methylation kinetics in synthetic DNA duplexes containing potential DNMT1 substrates was determined using a mass spectrometry based assay. Methylation rates decreased in the order mC&gt; hmC &gt; fC &gt; caC amd mC&gt; ethylC &gt; propylC. DNMT1 binding affinity for DNA duplexes containing mC, hmC, fC, caC, ethylC, propylC, and vinylC was elucidated using EMSA. Our results suggest that the reduced rates of methylation are associated with diminished specific binding of DNMT1 to structurally modified DNA duplexes. Computer modeling studies utilizing Schrödinger modeling package were used to provide a mechanistic understanding of the origins of altered methyltransferase activity in the presence of oxidized and expanded forms of 5-methylcytosine.<br/>

DNA methyltransferase DNMT1 activity in the presence of oxidized and extended forms of 5-methylcytosine

254th National Meeting (2017)

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