Crystal structure of the EcoKMcrA N-terminal domain (NEco): recognition of modified cytosine bases without flipping.

Anton Slyvka,Evelina Zagorskaitė,Matthias Bochtler,Giedrius Sasnauskas,Honorata Czapinska

doi:10.1093/nar/gkz1017

Abstract

EcoKMcrA from Escherichia coli restricts CpG methylated or hydroxymethylated DNA, and may act as a barrier against host DNA. The enzyme consists of a novel N-terminal specificity domain that we term NEco, and a C-terminal catalytic HNH domain. Here, we report that NEco and full-length EcoKMcrA specificities are consistent. NEco affinity to DNA increases more from hemi- to full-methylation than from non- to hemi-methylation, indicating cooperative binding of the methyl groups. We determined the crystal structures of NEco in complex with fully modified DNA containing three variants of the Y5mCGR EcoKMcrA target sequence: C5mCGG, T5mCGA and T5hmCGA. The structures explain the specificity for the two central base pairs and one of the flanking pairs. As predicted based on earlier biochemical experiments, NEco does not flip any DNA bases. The proximal and distal methyl groups are accommodated in separate pockets. Changes to either pocket reduce DNA binding by NEco and restriction by EcoKMcrA, confirming the relevance of the crystallographically observed binding mode in solution.

Highlights

Escherichia coli McrA (EcoKMcrA) has attracted interest for over 30 years as one of the natural barriers against genetic engineering of E. coli K strains [1,2,3]
The structure confirmed the presence of the predicted HNH nuclease domain at the C-terminus [7]. It showed that the N-terminal domain of EcoKMcrA, termed NEco in this work, is distantly similar to I-DreI, a dimeric meganuclease composed of I-DmoI and I-CreI homing endonucleases [8] and to MotA, a transcription factor from T4 phage [9]
Prior biochemical data on EcoKMcrA show that its N-terminal domain binds DNA in a methylation dependent manner [4]

Summary

Introduction

Escherichia coli McrA (EcoKMcrA) has attracted interest for over 30 years as one of the natural barriers against genetic engineering of E. coli K strains [1,2,3]. The restriction activity of EcoKMcrA on modified DNA is sequence context dependent. While binding of EcoKMcrA to modified DNA can be readily confirmed in the test tube, the cleavage of DNA by the enzyme has been elusive. The structure confirmed the presence of the predicted HNH nuclease domain at the C-terminus [7]. It showed that the N-terminal domain of EcoKMcrA, termed NEco in this work, is distantly similar to I-DreI, a dimeric meganuclease composed of I-DmoI and I-CreI homing endonucleases [8] and to MotA, a transcription factor from T4 phage [9]. It was not possible to deduce details of methyl- or hydroxymethyl recognition

Methods

Results

Conclusion