Protein-DNA selectivity is a central event in many biological processes. Type II restriction enzymes are ideal systems for studying selectivity, due to their high specificity and striking variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is 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 to design mutants with altered specificities. Our work is focused on the endonucleases Bam HI, Fok I, and Sfi I. We have determined structures of Bam HI at different stages of its catalytic pathway by X-ray crystallography. However, the critical structure that is missing for a complete understanding of specificity is that of a non-cognate DNA complex. Our specific goals are 1) to determine the structure of Bam HI bound to a non-cognate DNA site, 2) to determine the structure of the active site E113K mutant, and 3) to define the cleavage properties of the K205A mutant. Together, these studies will provide a complete, dynamic view of the sequence of events underlying sequence specific DNA cleavage. We have determined the structures of the complete Fok I endonuclease both with and without DNA. Fok I is an unusual bipartite enzyme that exists in solution as a monomer. A question that arises from our work is how the monomeric enzyme manages to cleave both strands? Our hypothesis is that Fok I dimerizes on DNA with the recognition domain of the second Fok I molecule accommodated on another DNA molecule (trans binding). Our specific goals are 1) to test this hypothesis by biophysical and biochemical methods, and 2) to use the bipartite nature of Fok I to create a novel hybrid enzyme capable of cleaving RNA at any pre-determined site. With Sfi I we now have cocrystals with DNA that diffract to 2.2 Angstrom units in resolution. Sfi I is a tetrameric enzyme that needs to interact with two DNA sites (either in cis or trans) in order to become activated for DNA cleavage. Our specific goals are 1) to determine the structure of the Sfi I/DNA complex using heavy atom derivatives, 2) to determine the structure of """"""""free"""""""" Sfi I using molecular replacement methods, and 3) to determine the structures of cleavage intermediates. The Sfi I reaction provides a system for understanding long-range interactions on DNA.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Lewis, Catherine D
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Mount Sinai School of Medicine
Schools of Medicine
New York
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Gupta, Yogesh K; Yang, Lin; Chan, Siu-Hong et al. (2012) Structural insights into the assembly and shape of Type III restriction-modification (R-M) EcoP15I complex by small-angle X-ray scattering. J Mol Biol 420:261-8
Vanamee, Eva Scheuring; Viadiu, Hector; Chan, Siu-Hong et al. (2011) Asymmetric DNA recognition by the OkrAI endonuclease, an isoschizomer of BamHI. Nucleic Acids Res 39:712-9
Vanamee, Eva Scheuring; Berriman, John; Aggarwal, Aneel K (2007) An EM view of the FokI synaptic complex by single particle analysis. J Mol Biol 370:207-12
Niv, Masha Y; Ripoll, Daniel R; Vila, Jorge A et al. (2007) Topology of Type II REases revisited;structural classes and the common conserved core. Nucleic Acids Res 35:2227-37
Townson, Sharon A; Samuelson, James C; Bao, Yongming et al. (2007) BstYI bound to noncognate DNA reveals a ""hemispecific"" complex: implications for DNA scanning. Structure 15:449-59
Vanamee, Eva Scheuring; Viadiu, Hector; Kucera, Rebecca et al. (2005) A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA. EMBO J 24:4198-208
Townson, Sharon A; Samuelson, James C; Xu, Shuang-Yong et al. (2005) Implications for switching restriction enzyme specificities from the structure of BstYI bound to a BglII DNA sequence. Structure 13:791-801
Townson, Sharon A; Samuelson, James C; Vanamee, Eva Scheuring et al. (2004) Crystal structure of BstYI at 1.85A resolution: a thermophilic restriction endonuclease with overlapping specificities to BamHI and BglII. J Mol Biol 338:725-33
Sun, Jian; Viadiu, Hector; Aggarwal, Aneel K et al. (2003) Energetic and structural considerations for the mechanism of protein sliding along DNA in the nonspecific BamHI-DNA complex. Biophys J 84:3317-25
Vanamee, Eva Scheuring; Hsieh, Pei chung; Zhu, Zhenyu et al. (2003) Glucocorticoid receptor-like Zn(Cys)4 motifs in BslI restriction endonuclease. J Mol Biol 334:595-603

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