Evolutionary Relationships, Design, and Biochemical Characterization of Homing Endonucleases

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Evolutionary Relationships, Design, and Biochemical Characterization of Homing Endonucleases

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Title: Evolutionary Relationships, Design, and Biochemical Characterization of Homing Endonucleases
Author: Taylor, Gregory Kevin
Abstract: Homing endonucleases, found in all forms of microbial life, facilitate the invasion of host genes often in concert with introns or inteins by generating double stranded breaks in conserved coding sequences. There are five homing endonuclease families distinct in their structural characteristics and each family appears to share a common ancestor with diverse host proteins of unrelated function. Such related proteins include restriction endonucleases, DNA mismatch repair proteins, transcription factors, four-way junction resolving enzymes, and colicins. Homing endonculeases are currently being computationally redesigned for applications in genome engineering and structures of three redesigned homing endonuclease variants are described. In these experiments, crystal structures uncovered unexpected shifts in the DNA backbone relative to the wild type endonucleases and have thus been informative in the redesign process. Recently, a sixth homing endonuclease family homologous to E. Coli DNA repair protein VSR was discovered. A series of biochemical and x-ray crystallographic experiments investigating binding specificity and catalytic mechanism of a representative family member I-Bth0305I are described. Finally, a database archiving experimentally characterized homing endonucleases and a web-base program supporting homing endonuclease target site search are discussed.
Description: Thesis (Ph.D.)--University of Washington, 2011
URI: http://hdl.handle.net/1773/19743
Author requested restriction: No embargo

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