Generation of a manganese specific restriction endonuclease with nicking activity

Abstract

A typical feature of type II restriction endonucleases (REases) is their obligate sequence specificity and requirement for Mg²⁺ during catalysis. R.KpnI is an exception. Unlike most other type II REases, the active site of this enzyme can accommodate Mg²⁺, Mn²⁺, Ca²⁺, or Zn²⁺ and cleave DNA. The enzyme belongs to the HNH superfamily of nucleases and is characterized by the presence of a ββα-Me finger motif. Residues D148, H149, and Q175 together form the HNH active site and are essential for Mg2+ binding and catalysis. The unique ability of the enzyme to cleave DNA in the presence of different metal ions is exploited to generate mutants that are specific to one particular metal ion. We describe the generation of a Mn²⁺-dependent sequence specific endonuclease, defective in DNA cleavage with Mg²⁺ and other divalent metal ions. In the engineered mutant, only Mn²⁺ is selectively bound at the active site, imparting Mn²⁺-mediated cleavage. The mutant is impaired in concerted double-stranded DNA cleavage, leading to accumulation of nicked intermediates. The nicking activity of the mutant enzyme is further enhanced by altered reaction conditions. The active site fluidity of REases allowing flexible accommodation of catalytic cofactors thus forms a basis for engineering selective metal ion-dependent REase additionally possessing nicking activity.