Introducing allosteric regulation into homing endonucleases via tryptophan modification

Abstract

Homing endonucleases (HEGs) are DNA cleaving enzymes present in microbial genomes that recognize unusually long DNA target sites (20–22 bp). Given their high specificity for DNA targets, these enzymes may be extremely useful genome editing reagents. Introducing allosteric control into these enzymes would enable them to have a broader applicability in genome editing approaches. Previous studies have shown that tryptophan residues near the active site of enzymes can play an important role in enzyme activity. Mutating these residues to glycine results in loss of enzymatic activity and can be rescued by the addition of indole in solution. However, this approach hasn't been demonstrated with DNA cleaving enzymes. The goal of this study is to test whether this approach can be used to introduce allosteric regulation into HEGs. We generated tryptophan to glycine single point mutations in I‐SmaMI, a homing endonuclease of the LAGLIDADG family, based on the structure of I‐SmaMI bound to DNA. We are expressing these proteins in bacteria and utilizing an in vitro cleavage assay to assess the level of activity of the mutants in the presence and absence of indole. These studies may have important genome editing applications and are likely to provide insight into the mechanism by which HEGs bind and cleave DNA.Support or Funding InformationMurdock Charitable TrustThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.