Indirect Readout and Specificity in DNA Binding Enzymes

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DESCRIPTION (provided by applicant): Recognition of DNA, both sequence and structure specific, is important in the healthy functioning of the cell, from replication, to transcription, to DNA repair. Failure of this process can lead to severe disease, including cancer. Both sequence and structure specific DNA recognition share a common mechanism involving the utilization of the intrinsic conformational energetics of DNA due, either to preferential base stacking interactions of different sequences, or to chemical and non-chemical modifications of the duplex structure. The project described herein is designed to investigate the molecular mechanisms of sequence specificity, and the relaxation of this specificity, in DNA binding enzymes. The use of enzymes in such studies is advantageous over the use of non-enzymatic DNA binding proteins, as thermodynamic parameters can be extracted more faithfully as a result of the greater structural stringency of the transition state relative to the ground state complex. Rather than focusing on direct protein-DNA interactions, the investigations address subtle and more complex mechanisms of recognition, including the utilization of sequence dependent conformational preferences of the DNA and conformational changes occurring within a protein as a result of allosteric modulation. Selected type II restriction endonucleases will be used in the study, due to their unparalleled specificity, activity, diversity, and experimental practicality. A combination of three dimensional structure determinations using x-ray crystallography, as well as kinetic and binding affinity measurements, will be used in the investigations.