Discovery of 5mYOX-associated C5-cytosine hypermodifying enzymes

We recently illustrated that 5mYOXs from viral metagenomes exist within biosynthetic gene clusters (BGCs) with functions related to DNA hypermodification (Burke, E. J. et al. (2021) Proc. Natl. Acad. Sci. U S A., 118(26):e2026742118). Enzymes that traditionally cluster with 5mYOXs either install the 5mC substrate precursor for 5mYOX activity (DNA C5-cytosine methyltransferases) or chemically derivatize the 5hmC product of 5mYOX to generate hypermodified bases. 5mYOX-associated hypermodification enzymes identified by our lab include DNA glycosyltransferases, kinases, and amino acid transferases. We are developing and optimizing high-throughput platforms for identifying the DNA natural products that result from the activities of phage-encoded BGCs, characterization of the hypermodifying enzyme chemistries, and mapping the modification loci in the genome. Additionally, we are collaborating with other NEB research labs to develop innovative reverse genetic systems to explore the biological roles of DNA hypermodifications in phage infection cycles. Our discovery platforms have revealed diverse new base modifications from phage BGCs that we speculate would be useful in emerging applications such as next-generation sequencing technologies, aptamer chemistry, and DNA-based therapeutics.