Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA.

Liudmila A Abrosimova,Tatiana S Oretskaya,Tatiana A Perevyazova,Natalia A Astafurova,Elena A Kubareva,Andrey S Karpov,Nikita A Kuznetsov,Anastasiia R Samsonova,Olga S Fedorova

doi:10.3390/biom11101420

Abstract

Nicking endonucleases (NEs) are enzymes that incise only one strand of the duplex to produce a DNA molecule that is ‘nicked’ rather than cleaved in two. Since these precision tools are used in genetic engineering and genome editing, information about their mechanism of action at all stages of DNA recognition and phosphodiester bond hydrolysis is essential. For the first time, fast kinetics of the Nt.BspD6I interaction with DNA were studied by the stopped-flow technique, and changes of optical characteristics were registered for the enzyme or DNA molecules. The role of divalent metal cations was estimated at all steps of Nt.BspD6I–DNA complex formation. It was demonstrated that divalent metal ions are not required for the formation of a non-specific complex of the protein with DNA. Nt.BspD6I bound five-fold more efficiently to its recognition site in DNA than to a random DNA. DNA bending was confirmed during the specific binding of Nt.BspD6I to a substrate. The optimal size of Nt.BspD6I’s binding site in DNA as determined in this work should be taken into account in methods of detection of nucleic acid sequences and/or even various base modifications by means of NEs.

Highlights

Nicking endonucleases (NEs) are enzymes that recognise specific sequences in doublestranded DNA, but unlike restriction endonucleases (REs, Rs), NEs cleave only one DNA strand at a certain position relative to the recognition site
We showed that the minimal kinetic scheme of the Nt.BspD6I interaction with DNA in the presence of Mg2+ includes several stages: non-specific DNA binding, a search for the recognition site, the formation of a specific enzyme–substrate complex accompanied by DNA bending, catalysis, and dissociation of the enzyme from the product
We investigated the influence of the length of the DNA sequences flanking the recognition site and the impact of two divalent cations acting as cofactors on the kinetics of the Nt.BspD6I–DNA interaction

Summary

Introduction

Nicking endonucleases (NEs) are enzymes that recognise specific sequences in doublestranded DNA (dsDNA), but unlike restriction endonucleases (REs, Rs), NEs cleave only one DNA strand at a certain position relative to the recognition site. The first NE was discovered in 1996 in the thermophilic microorganism Bacillus stearothermophilus SE-589 [1]. According to the REBASE (http://rebase.neb.com/cgi-bin/azlist?nick, accessed on 21 September 2021), more than 14,000 NEs are known (including putative enzymes). Most of the characterised NEs are from bacteria of the genus Bacillus. The NEs that catalyse the cleavage of the top or bottom DNA strand in the duplex (50 →30 /30 →50 ) are denoted as Nt (‘nicking top’) and Nb (‘nicking bottom’), respectively

Objectives

Methods

Results

Conclusion