Protein-DNA recognition is a fundamental event in many biological processes. Type II restriction enzymes are ideal systems for studying protein-DNA interactions because of their high sequence specificity and striking variety. Surprisingly, restriction enzymes show little protein sequence similarity. The cleavage of DNA by type II restriction enzymes is also highly sequence specific, occuring only when all the sequence specific protein-DNA interactions have formed. An understanding of sequence specific cleavage is also relevant to proteins mediating site-specific recombination, and DNA repair by excision. The long term goals of this project are to understand the mechanisms by which type II restriction enzymes recognize and cleave DNA, and the design of mutants with altered DNA specificities. To accomplish these goals, the specific aims of this proposal are: 1) To determine the structure of BamHI, one of the best characterized restriction enzymes, by X-ray crystallography to a resolution of 2.3Ao. We already have highly stable crystals of the enzyme in two forms. 2) To determine the structure of BamHI complexed with a fragment of DNA encompassing its recognition site. We have shown that BamHI is capable of making a stable complex with a fragment of DNA, and have initiated efforts to obtain cocrystals. An important part of our strategy will be to search crystallization conditions with different length DNA fragments. 3) To determine the structure of BamHI complexed to various cleaved DNA products, representing various intermediate states in the catalytic pathway. Crystals for these studies will be obtained by soaking Mg2+ into BamHI-intact DNA cocrystals, and standard crystallographic and Laue diffraction techniques will be applied to obtain the structures. 4) To study site-directed and random mutants of BamHI by difference Fourier maps, in order to test ideas on recognition and cleavage, and to design mutants of BamHI with altered DNA specificities. 5) To determine the structures of BamHI methylase and BamHI isoschizomers, in order to understand fully every facet of the recognition process. 6) To determine the structure of TaqI and other restriction endonucleases. Crystals of TaqI diffracting to 8.0Ao have been obtained, but efforts will continue to obtain better ordered crystals.
Showing the most recent 10 out of 22 publications
