Thermodynamic, Kinetic, and Structural Basis for the Relaxed DNA Sequence Specificity of "Promiscuous" Mutant EcoRI Endonucleases

Promiscuous mutant EcoRI endonucleases produce lethal to sub-lethal effects because they cleave E. coli DNA despite the presence of the EcoRI methylase. Three promiscuous mutant forms, Ala138Thr, Glu192Lys and His114Tyr, have been characterized with respect to their binding affinities and first-order cleavage rate constants towards the three classes of DNA sites: specific, miscognate (EcoRI*) and nonspecific. We have made the unanticipated and counterintuitive observations that the mutant endonucleases that exhibit relaxed specificity in vivo nevertheless bind more tightly than the wild-type enzyme to the specific recognition sequence in vitro and show even greater preference for binding to the cognate GAATTC site over miscognate sites. Binding preference for EcoRI* over nonspecific DNA is also improved. The mutant enzymes cleave the cognate site GAATTC at a normal rate, but cleave EcoRI* sites faster than does the wild-type enzyme. Thus, the mutant enzymes use two mechanisms to partially bypass the multiple fail-safe mechanisms that protect against cleavage of genomic DNA in cells carrying the wild-type EcoRI restriction-modification system: (a) Binding to EcoRI* sites is more probable than for wild-type enzyme because nonspecific DNA is less effective as a competitive inhibitor; (b) The combination of increased affinity and faster cleavage at EcoRI* sites makes double-strand cleavage of these sites a more probable outcome than it is for the wild-type enzyme. The crystal structure of the A138T "promiscuous" mutant enzyme in complex with specific DNA shows reveals no changes in protein contacts to the bases of the GAATTC site relative to the wild-type complex; however, there are changes in water-mediated contacts between the enzyme and flanking bases, and changes in protein-phosphate contacts. These observations lead us to hypothesize that the improved specific DNA binding of the A138T enzyme relative to the wild-type enzyme is not attributable to a single new protein DNA contact, but rather the distributed effect of the improved complementarity between the mutant and the flanking bases, as well as the optimization of several phosphate contacts. The aggregate of biochemical data presented in this thesis leads us to propose a model where the A138T miscognate DNA binding ensemble (for a subset of miscognate sites) is partitioned more towards complexes that are on the path to the transition state than wild-type miscognate DNA binding ensembles; the mutant accomplishes this by forming 'specific-like' phosphate contacts to these sites, which in turn stabilize the DNA distortion that is critical for efficient cleavage. Given the proximity of amino acid 138 to the residues which make contacts to the DNA phosphates in the specific complex, we hypothesize that the A138T mutation has an effect on the structure and/or dynamics of the "arm" protein segment (contains residues contacting the DNA backbone) such that it is more adaptable, resulting in formation of functional phosphate contacts to a broader range of DNA substrates