Investigating The DNA Binding Constraints Of The I‐Sob2593 Meganuclease

Meganucleases are DNA cleaving enzymes present in microbial genomes that are considered “selfish” elements because they facilitate their spread to other genomes. They are prime candidates as genome editing reagents because they recognize unusually long DNA sequences (18–22 bp). However, redirecting their specificity towards novel targets remains a challenge since the same surface of the protein mediates DNA and binding and cleavage. A recent study of unicellular green algae identified the widespread insertion of meganuclease homologs at conserved sites within mitochondrial and chloroplast genomes. Analysis of a subset of these homologs demonstrated that the enzymes are capable of cleaving an identical (or nearly identical) DNA target sequence despite having limited sequence identity (34–60%). These results suggest that the enzymes have evolved unique solutions to recognizing a common DNA target. We have focused on characterizing the meganuclease I‐Sob2593. We have solved the x‐ray structure of I‐SobI2593 bound to DNA, allowing molecular level comparisons of binding mechanics to a previously solved meganuclease, I‐CreI. We will also determine I‐Sob2593's tolerance to polymorphism along its target sequence using in vitro cleavage assays. In future experiments we will expand this analysis to other homologs of I‐Sob2593. Completion of these experiments will give insight into the rules that govern DNA recognition by these enzymes and will serve as a starting point to redirect their specificity towards desired DNA targets. Support or Funding Information Murdock Trust