Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain.

Shuang-Yong Xu

doi:10.3389/fmicb.2021.787073

Abstract

Strand-specific DNA nicking endonucleases (NEases) typically nick 3–7 bp sites. Our goal is to engineer infrequent NEase with a >8 bp recognition sequence. A BamHI catalytic-deficient mutant D94N/E113K was constructed, purified, and shown to bind and protect the GGATCC site from BamHI restriction. The mutant was fused to a 76-amino acid (aa) DNA nicking domain of phage Gamma HNH (gHNH) NEase. The chimeric enzyme was purified, and it was shown to nick downstream of a composite site 5′ GGATCC-N(4-6)-AC↑CGR 3′ (R, A, or G) or to nick both sides of BamHI site at the composite site 5′ CCG↓GT-N5-GGATCC-N5-AC↑CGG 3′ (the down arrow ↓ indicates the strand shown is nicked; the up arrow↑indicates the bottom strand is nicked). Due to the attenuated activity of the small nicking domain, the fusion nickase is active in the presence of Mn2+ or Ni2+, and it has low activity in Mg2+ buffer. This work provided a proof-of-concept experiment in which a chimeric NEase could be engineered utilizing the binding specificity of a Type II restriction endonucleases (REases) in fusion with a nicking domain to generate infrequent nickase, which bridges the gap between natural REases and homing endonucleases. The engineered chimeric NEase provided a framework for further optimization in molecular diagnostic applications.

Highlights

In nature, DNA nicking endonucleases (NEases) are mainly involved in DNA restriction, rolling circle replication, F plasmid transfer, and DNA damage repair (Zheleznaya et al, 2009; Chan et al, 2011; Xu, 2015)
The activity on the Gamma HNH (gHNH) nicking site ACCGR or its variants alone is rather low, and no doublets were detected in run-off sequencing of ACCGR sites further downstream or upstream under limited digestion, which is likely due to the attenuated activity of the small nicking domain that requires a strong DNA binding partner to anchor onto DNA
The nicking site recognition could be relaxed to nick star sites (ASYSR) in Mn2+ buffer, which is consistent with the star activity of the full-length gHNH endonuclease in Mn2+ buffer (1–2 base off from ACCGR sites) (Xu and Gupta, 2013)

Summary

Introduction

DNA nicking endonucleases (NEases) are mainly involved in DNA restriction, rolling circle replication, F plasmid transfer, and DNA damage repair (Zheleznaya et al, 2009; Chan et al, 2011; Xu, 2015). The restriction endonuclease (REase)-derived nicking enzymes typically nick DNA target sites in 3–7 bp, and homing NEases recognition sequences are usually more than 16 bp (Chan et al, 2011). To expand the DNA target sequence range, we propose to utilize the DNA binding specificities of Type II REases (3–8 bp) that are cleavage-deficient with mutations in the catalytic site and fusion of the mutant enzymes to a DNA nicking domain [e.g., phage Gamma HNH (gHNH) nicking endonuclease, gHNH, 3–5 bp recognition (Xu and Gupta, 2013)]. When a REase binding specificity for 7–8 bp target is fused to a gHNH nicking domain of 5 bp (e.g., ACCGR, complementary strand YCGGT, R = A or G, Y = C or T), it is possible to generate an Nicking Endonuclease With BamHI*-gHNH Fusion infrequent nicking enzyme with 12–13 bp composite sites. It is shown that the minimal DNA nicking domain of the gHNH endonuclease contains only 76 amino acid (aa) residues and required Mn2+ for stimulated nicking activities (Xu and Gupta, 2013)

Objectives

Methods

Results

Conclusion