Unique Solutions to a Common Problem: Understanding How I‐CreI Homologs Recognize the Same DNA Target Sequence

Abstract

Homing endonucleases (or “meganucleases”) are DNA cleaving enzymes that are widely present in microbial genomes. They are known as “selfish elements” for their ability to cleave their DNA target site within related species and insert themselves into the genome via homologous recombination. We studied a family of related LAGLIDADG homing endonucleases (“I‐Cre1 homologs”) which have been identified within the large ribosomal subunit gene of the chloroplast genome of numerous unicellular green algae. Despite significant divergence at the amino acid level (40–60% identity), these enzymes have retained the ability to cleave nearly identical DNA targets. For this reason, this enzyme family is a great case study to understand protein‐DNA interactions. Our goal was to investigate the unique ways in which a subset of I‐Cre1 homologs cleave their DNA targets. We used X‐ray crystallography to solve the molecular structures of three homologs bound to DNA then constructed contact maps for all of the individual protein‐DNA contacts being made. We also generated cleavage specificity profiles by performing DNA cleavage assays using one‐off mutated substrates for four enzymes (Col2593c, Sob2593c, Cre2593c and Hla2593c). The specificity profiles and DNA‐protein contact maps for the enzymes were analyzed alongside a multiple sequence alignment to identify how key contacts confer regions of specificity and flexibility. These conclusions allow for a detailed analysis of the unique ways each protein recognizes and cleaves their common DNA target.Support or Funding InformationMurdock Charitable Trust (research)