Modifying a covarying protein–DNA interaction changes substrate preference of a site-specific endonuclease

Abstract

Identifying and validating intermolecular covariation between proteins and their DNA-binding sites can provide insights into mechanisms that regulate selectivity and starting points for engineering new specificity. LAGLIDADG homing endonucleases (meganucleases) can be engineered to bind non-native target sites for gene-editing applications, but not all redesigns successfully reprogram specificity. To gain a global overview of residues that influence meganuclease specificity, we used information theory to identify protein–DNA covariation. Directed evolution experiments of one predicted pair, 227/+3, revealed variants with surprising shifts in I-OnuI substrate preference at the central 4 bases where cleavage occurs. Structural studies showed significant remodeling distant from the covarying position, including restructuring of an inter-hairpin loop, DNA distortions near the scissile phosphates, and new base-specific contacts. Our findings are consistent with a model whereby the functional impacts of covariation can be indirectly propagated to neighboring residues outside of direct contact range, allowing meganucleases to adapt to target site variation and indirectly expand the sequence space accessible for cleavage. We suggest that some engineered meganucleases may have unexpected cleavage profiles that were not rationally incorporated during the design process.

Optimization of Protein Thermostability and Exploitation of Recognition Behavior to Engineer Altered Protein-DNA Recognition

“…Adding additional layers of complexity to the engineering process are the observations that (1) for many LAGLIDADG protein-DNA complexes, overall binding affinity is dominated by formation of contacts across half of the protein-DNA interface (Thyme et al, 2009), (2) that distal mutations can unexpectedly alter specificity elsewhere in the protein-DNA interface (Laforet et al 2019), and (3) that a frequently observed amino acid substitution in the LA-GLIDADG protein family (corresponding to E178D in I-OnuI) that increases tolerance for (and activity against) target sites with altered central base pairs (McMurrough et al, 2018;Takeuchi et al, 2011) is a key element in the engineering process.…”

Intein-based thermoregulated meganucleases for containment of genetic material

“…Primers were ordered from Integrated DNA Technologies. Wild-type I-OnuI was previously cloned between the NcoI and NotI sites in pProEX-HT-a (Invitrogen and Life Technologies) and pEndo backbone ( 38 , 39 ).…”

Gene Editing in Pluripotent Stem Cells and Their Derived Organoids

“…Meganucleases (MegNs) rely on the length of the target sequence and the structure of the DNA contact surface to specifically, accurately, and effectively identify the target. The mechanism of DNA recognition by MegNs involves binding patterns of protein side chains and nucleotide bases [ 15 ], deformation of groove dimensions, electrostatic distribution of the molecular surface, and additional contacts within and near the minor groove [ 16 ]. Binding affinity and cleavage activity sometimes have different efficiencies.…”