# Conversion of the CG specific M.MpeI DNA methyltransferase into an enzyme predominantly methylating CCA and CCC sites

## TL;DR

Scientists modified a DNA methylating enzyme to change its target from CG to CCA and CCC sites using mutagenesis and selection.

## Contribution

A new DNA methyltransferase variant with specificity for CCA and CCC sites was created from a CG-specific enzyme.

## Key findings

- M.MpeI variants showed increased activity on CC and CA sites after mutagenesis.
- A final variant predominantly methylates CC dinucleotides with a strong preference for CCA and CCC sites.
- The mutant enzyme has a deletion and three substitutions in its target recognition domain.

## Abstract

We used structure guided mutagenesis and directed enzyme evolution to alter the specificity of the CG specific bacterial DNA (cytosine-5) methyltransferase M.MpeI. Methylation specificity of the M.MpeI variants was characterized by digestions with methylation sensitive restriction enzymes and by measuring incorporation of tritiated methyl groups into double-stranded oligonucleotides containing single CC, CG, CA or CT sites. Site specific mutagenesis steps designed to disrupt the specific contacts between the enzyme and the non-substrate base pair of the target sequence (5′-CG/5′-CG) yielded M.MpeI variants with varying levels of CG specific and increasing levels of CA and CC specific MTase activity. Subsequent random mutagenesis of the target recognizing domain coupled with selection for non-CG specific methylation yielded a variant, which predominantly methylates CC dinucleotides, has very low activity on CG and CA sites, and no activity on CT sites. This M.MpeI variant contains a one amino acid deletion (ΔA323) and three substitutions (N324G, R326G and E305N) in the target recognition domain. The mutant enzyme has very strong preference for A and C in the 3′ flanking position making it a CCA and CCC specific DNA methyltransferase.

Graphical Abstract