STRUCTURAL MECHANISMS OF DNA RECOGNITION AND CLEAVAGE BY SEQUENCE SPECIFIC DNA B

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal includes studies aimed at understanding, via structural means, of several aspects of DNA recognition and cleavage by model enzymes of the type II restriction endonuclease class. First, the role of indirect readout will be investigated by structurally characterizing mutants of HincII possessing altered sequence specificity, where the distance between the site of mutation and the DNA where specificity has been altered is large. Second, as nearly all DNA endonucleases, including those involved in critical cellular functions such as replication, recombination, and repair, utilize divalent cation dependent catalysis, the divalent cation dependent stereochemical cleavage mechanism of DNA cleavage by HincII will be investigated. Finally, enzymes exist which possess altered sequence specificity upon cleavage of their normal target sequence in DNA. The de Novo structure of one such enzyme, SgrAI, bound to DNA will be determined.