The detailed structural and energetic origins of sequence-specificity in the recognition and modification of DNA will be investigated using prokaryotic restriction endonucleases and modification methylases as experimental systems. First, the specificity-determining induced fit conformational change in the best-studied EcoRV endonuclease will be examined using stopped-flow fluorescence techniques, chemical quench-flow kinetics, and Xray crystallography. These approaches will be applied to the study of noncognate complexes and to enzyme variants, to test current hypotheses regarding the origins of sequence selectivity. The mechanism of DNA bending by this enzyme will also be tested by modifications to both enzyme and DNA, coupled to functional and structural analyses. Next, the diversity of metal-dependent cleavage chemistries in three other structurally well-characterized type II restriction endonucleases will be studied by enzymological techniques. {{Finally, we shall extend our inquiry into the induced-fit origins of sequence selectivity by exploring how the """"""""base-flipping"""""""" structural transitions in both adenine and cytosine DNA methylases play crucial roles in mediating the approach to the transition state in cognate and noncognate complexes.}} Elucidation of how DNA sequence recognition is manifested through induced-fit conformational rearrangements in the restriction endonuclease and methylase families will be relevant to understanding this phenomenon in a variety of nucleic-acid modifying enzymes including recombinases, topoisomerases, and enzymes of DNA repair and RNA modification pathways. Further, a clearer appreciation of the detailed structural and energetic basis for specificity and rate enhancement forms an essential basis for the rational engineering of enzymes with new specificities. Since restriction endonucleases and DNA methylases are an essential part of the recombinant DNA technology that underlies all areas of biological research, this is a prospect of substantial importance to the development of therapies for many human diseases.
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