Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA.

Michal Pastor,Honorata Czapinska,Thomas Lutz,Katarzyna Krakowska,Shuang-Yong Xu,Igor Helbrecht,Matthias Bochtler

doi:10.1093/nar/gkaa1218

Abstract

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.

Highlights

Most restriction modification systems use methylation as a mark of ‘self’ and target non-modified DNA
Modification dependent restriction endonucleases (MDREs) that do not depend on nucleoside triphosphates (NTPs) are typically encoded by single open reading frames, and are modular proteins that consist of separate modification-sensing and nuclease domains
Crystals without DNA, with single-stranded DNA (ssDNA), and with double-stranded DNA (dsDNA) grew in different space groups, the protein conformation was remarkably similar in all structures, with the exception of two loops in proximity of the DNA

Summary

Introduction

Most restriction modification systems use methylation as a mark of ‘self’ and target non-modified DNA. Such systems are typically sequence specific and include a methyltransferase (MTase) that protects DNA by methylation in the context relevant for cleavage. Perhaps in response to the wide-spread occurrence of ‘canonical’ restriction systems, many phages have modified bases in their genomes that protect against this defense. Modification dependent restriction endonucleases (MDREs) target DNA that is modified, and are non-toxic to a host with non-modified DNA. They are ‘orphan’ in the sense that they come without accompanying MTases, but unlike other restriction modification systems, they are often present in defense islands.

Methods

Results

Conclusion