Structure determination, thermodynamic characterization, and computational redesign of the rare-cutting restriction endonuclease NotI

NotI is a rare-cutting restriction enzyme which cleaves the 8-base pair recognition sequence 5'-GCGGCCGC-3'. Due to its rare-cutting cleavage activity, NotI is commonly used as an enzymatic reagent to generate large genomic fragments in restriction landmark genomic scanning (RLGS), a technique used for wide-scale characterization of genomic alterations. The GC composition of the enzyme's target DNA sequence makes NotI especially well-suited for the study of DNA methylation patterns using this technique. The crystal structure of NotI has been solved in the presence and absence of its DNA substrate to 2.5A and 2.8A resolution, respectively. The enzyme's endonuclease core fold is augmented with a unique metal-binding fold occupied by a single iron atom in a tetrahedral Fe-Cys4 motif. The iron-binding domain helps position adjacent structural elements critical for proper DNA recognition, and it appears to serve a purely structural role. The active site of NotI contains the PD...(D/E)xK nuclease motif and an architecture appropriate for the hydrolysis of DNA phosphodiester bonds via a two-metal ion mechanism. While recognition of the central six basepairs of the NotI DNA target site is accomplished by a saturated hydrogen bond network typical of restriction enzymes, the most peripheral basepair in each half-site is engaged by only a single direct contact in the major groove---possibly reflecting reduced pressure to maintain specific recognition at those positions. Binding and cleavage of DNA by NotI is blocked by methylation of cytosines at positions 2 and 6 of its recognition sequence. Structure-based computational design methods were applied towards the redesign of NotI for binding of methylated DNA target sites. Thermodynamic characterization by isothermal titration calorimetry (ITC) has shown that NotI binds DNA using an "enthalpy-entropy compensation" commonly observed for specific DNA-binding proteins.