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DNA methylation of cytosine in eukaryotic cells is a common epigenetic modification, which plays an important role in gene expression and thus affects various cellular processes like development and carcinogenesis. The occurrence of 5-methyl-2'-deoxycytosine (5mC) as well as the distribution pattern of this epigenetic marker were shown to be crucial for gene regulation and can serve as important biomarkers for diagnostics. DNA polymerases distinguish little, if any, between incorporation opposite C and 5mC, which is not surprising since the site of methylation is not involved in Watson-Crick recognition. Here, we describe the development of a DNA polymerase variant that incorporates the canonical 2'-deoxyguanosine 5'-monophosphate (dGMP) opposite C with higher efficiency compared to 5mC. The variant of Thermococcus kodakaraensis (KOD) exo- DNA polymerase was discovered by screening mutant libraries that were built by rational design. We discovered that an amino acid substitution at a single site that does not directly interact with the templating nucleobase, may alter the ability of the DNA polymerase in processing C in comparison to 5mC. Employing these findings in combination with a nucleotide, which is fluorescently labeled at the terminal phosphate, indicates the potential use of the mutant DNA polymerase in the detection of 5mC.

5-methylcytosine-sensitive variants ofThermococcus kodakaraensisDNA polymerase