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

SUBMITTER: Laforet M 

PROVIDER: S-EPMC6847045 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature