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Evolutionary Relationships, Design, and Biochemical Characterization of Homing Endonucleases

Taylor, Gregory (2011) Evolutionary Relationships, Design, and Biochemical Characterization of Homing Endonucleases. PhD thesis, Fred Hutchinson Cancer Research Center / University of Washington.

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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.

Item Type: Thesis (PhD)
Subjects: Cellular and Organismal Processes > Evolution
Molecules > Proteins > Enzymes
Depositing User: Craig Johansen
Date Deposited: 01 Mar 2012 18:34
Last Modified: 01 Mar 2012 18:34
URI: http://authors.fhcrc.org/id/eprint/541

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