Mutational analysis of the Clu2593c homing endonuclease reveals specific residues required for DNA binding and cleavage.

Abstract

Homing endonucleases (HEGs) are selfish genetic elements that recognize and cleave long DNA target sequences (18–22 basepairs) within an organism's genome. This cleavage induces homology‐directed repair using the HEG‐encoding gene as a template, facilitating its lateral transmission. The I‐CreI family of HEGs is present within organellar genomes of diverse single‐celled algae species and is incorporated at identical positions within the large ribosomal subunit gene. Homologs of this family, which share 35–70% pairwise amino acid identity, have evolved to recognize nearly identical target sites while tolerating sequence polymorphisms along the DNA target site. Although the I‐CreI and I‐CvuI enzymes have been well characterized, the mechanisms by which other homologs of this family achieve target sequence recognition are not well understood. In order to explore the structure‐function relationship of the I‐CreI family, we carried out a detailed molecular‐level analysis of the I‐CreI homolog Clu2593c (57% sequence identity). Based on a co‐crystal structure of WT Clu2593c bound to DNA, we generated 12 single‐residue mutations at positions in the enzyme‐DNA interface. We then tested the functionality of the bacterially‐expressed and purified mutant proteins using in vitro binding and cleavage assays. Additionally, we solved co‐crystal structures of a subset of the mutants bound to DNA. Our analyses revealed that several of the mutations behaved in a similar manner to analogous mutations in I‐CreI, whereas others indicated divergent mechanisms of DNA recognition. These results highlight that homologs of the I‐CreI family have evolved distinct solutions to recognizing a shared DNA target.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.