Discovery And Application Of Cytosine Carboxymethylation

Evolution molds biological function. DNA, the scaffold upon which natural evolution progresses, is composed of four nucleobases: adenine, cytosine, guanine, and thymine. As the genome propagates across generations of replication, these nucleobases must find a way to balance their primary function of maintaining trans-generational fidelity while still leaving room for purposeful mutation and adaptation. Within generations, additional opportunities to encode new functions exist through the intentional chemical modification of DNA. In particular, DNA methylation, found within Cytosine-Guanine (CpG) dinucleotides, represents a canonical “epigenetic” mark, whereby covalent modification to cytosine perturbs gene expression. In our attempt to understand the catalytic determinants of 5-methylcytosine (5mC) generation by DNA methyltransferase (MTase) enzymes, we created an unexpected and previously unknown DNA modification, 5-carboxymethylcytosine (5cxmC). We show that only a single point mutation in the active site is responsible for conferring neomorphic activity on this enzyme family, effectively turning DNA MTases into DNA carboxymethyltransferases (CxMTases). In E. coli, these CxMTases can shunt the sparse but natural metabolite carboxy-S-adenosyl-L-Methionine (CxSAM) to be directly used as a substate to make 5cxmC in genomic DNA. Our discovery of this new CxMTase/CxSAM, enzyme/substrate pair further enabled the development a new biotechnology, termed Direct-Methylation Sequencing (DM-Seq), which can directly localize 5mCpGs at single base resolution using limited DNA input. Our findings advance both synthetic biology and biotechnology, whereby our laboratory-based evolution characterizes the biochemical circumstances limiting the natural evolution of DNA modifications and simultaneously reveal the power of structure-guided protein engineering in unlocking a transformative epigenetic sequencing technology.