On the role of steric clashes in methylation control of restriction endonuclease activity.

Karolina Mierzejewska,Matthias Bochtler,Honorata Czapinska

doi:10.1093/nar/gkv1341

Abstract

Restriction-modification systems digest non-methylated invading DNA, while protecting host DNA against the endonuclease activity by methylation. It is widely believed that the methylated DNA would not ‘fit’ into the binding site of the endonuclease in the productive orientation, and thus steric clashes should account for most of the protection. We test this concept statistically by grafting methyl groups in silico onto non-methylated DNA in co-crystal structures with restriction endonucleases. Clash scores are significantly higher for protective than non-protective methylation (P < 0.05% according to the Wilcoxon rank sum test). Structural data alone are sufficient to distinguish between protective and non-protective DNA methylation with 90% confidence and decision thresholds of 1.1 Å and 48 Å3 for the most severe distance-based and cumulative volume-based clash with the protein, respectively (0.1 Å was deducted from each interatomic distance to allow for coordinate errors). The most severe clashes are more pronounced for protective methyl groups attached to the nitrogen atoms (N6-methyladenines and N4-methylcytosines) than for C5-methyl groups on cytosines. Cumulative clashes are comparable for all three types of protective methylation.

Highlights

Restriction-modification (RM) systems consist of endonucleases that cleave invading unmodified DNA and their cognate methyltransferases of matching or slightly broader specificity, which protect genomic DNA of the host from this fate
Structures of type II restriction endonucleases with DNA bound in a productive orientation were obtained from the Protein Data Bank (PDB) [3]
The statistical analysis presented in this work demonstrates that methyl groups protecting against restriction endonuclease cleavage have more steric conflict with the enzymes than those that do not block the reaction

Summary

Introduction

Restriction-modification (RM) systems consist of endonucleases that cleave invading unmodified DNA and their cognate methyltransferases of matching or slightly broader specificity, which protect genomic DNA of the host from this fate. Hemimethylated DNA arises after semiconservative replication of fully methylated DNA and is the preferred substrate for the RM methyltransferases. In vitro they modify non-methylated DNA which tends to blur the distinction between host and invading DNA. In order to avoid damage to the host genome during DNA replication, hemimethylation is typically sufficient to prevent restrictase-mediated DNA cleavage. Experimental data on the influence of methyl groups on the susceptibility of DNA to cleavage by restriction endonucleases have been collected in the REBASE database (http: //rebase.neb.com) [1]

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Results

Conclusion