The ultimate goal of this research is to elucidate molecular mechanisms of sequence-specific DNA-protein interactions. This project proposes to continue structure-function analysis of EcoRI endonuclease, a restriction enzyme which recognizes the hexanucleotide GAATTC. The proposal focuses on the recognition mechanism. the structure of the endonuclease-- TCGCGAATTCGCG complex has been determined to 3 angstroms resolution. Refinement to better than 2.3 A resolution is in progress, as are structure determinations for related forms; including enzyme-product cocrystals obtained by soaking enzyme-substrate cocrytals in MgII and Mn II. The diffraction pattern is retained during the cleavage reaction; one of the project goals is to follow the reaction via time-resolved crystallography. Additional structure determinations will b initiated for cocrystals containing selected site-directed mutations of the endonuclease and/or altered oligonucleotides. Efforts will be made to cocrystalize the RsrI endonuclease, an isoschizmer of EcoRI. A major goal of these structure determinations is to derive a """"""""comparative anatomy"""""""" of the endonuclease-DNA system that can be correlated with biological function. Ultimately, these correlations will have to be quantitative; a long range goal is to connect the structural observations with relative binding free energies, cleavage rates and other experimentally observable quantities. The first steps in this direction are to carry out computer simulations on the structures determined here. Current simulations are focused on the energetics of DNA kinking.

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National Institute of General Medical Sciences (NIGMS)
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Molecular and Cellular Biophysics Study Section (BBCA)
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University of Pittsburgh
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