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

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.

SUBMITTER: Xu SY 

PROVIDER: S-EPMC8845596 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature