# Systematic analysis of specificities and flanking sequence preferences of bacterial DNA-(cytosine C5)-methyltransferases reveals mechanisms of enzyme- and sequence-specific DNA readout

## TL;DR

This study explores how bacterial enzymes that methylate DNA recognize specific sequences and how their activity is influenced by surrounding DNA sequences.

## Contribution

The study reveals how DNA methylation specificity and flanking sequence preferences are modulated by enzyme-DNA interactions and DNA conformation.

## Key findings

- Bacterial MTases show high (>1000-fold) target sequence specificity and strong flanking sequence preferences (∼100-fold).
- Mutations in DNA-contacting amino acids alter both local and global enzyme specificity and flanking sequence preferences.
- Flanking sequence preferences are likely due to sequence-dependent modulation of DNA conformation.

## Abstract

DNA-(cytosine C5)-methyltransferases (MTases) represent a large group of evolutionary related enzymes with specific DNA interaction. We systematically investigated the specificity and flanking sequence preferences of six bacterial enzymes of this class and many MTase mutants. We observed high (>1000-fold) target sequence specificity reflecting strong evolutionary pressure against unspecific DNA methylation. Strong flanking sequence preferences (∼100-fold) were observed which changed for methylation of near-cognate sites suggesting that the DNA structures in the transition states of the methylation of these sites differ. Mutation of amino acids involved in DNA contacts led to local changes of specificity and flanking sequence preferences, but also global effects indicating that larger conformational changes occur upon transition state formation. Based on these findings, we conclude that the transition state of the DNA methylation reaction precedes the covalent enzyme–DNA complex conformations with flipped target base that are resolved in structural studies. Moreover, our data suggest that alternative catalytically active conformations exist whose occupancy is modulated by enzyme–DNA contacts. Sequence dependent DNA shape analyses suggest that MTase flanking sequence preferences are caused by flanking sequence dependent modulation of the DNA conformation. Likely, many of these findings are transferable to other DNA MTases and DNA interacting proteins.

Graphical Abstract