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dc.contributor.authorGalburt, Eric Aen_US
dc.date.accessioned2009-10-06T23:12:18Z
dc.date.available2009-10-06T23:12:18Z
dc.date.issued2002en_US
dc.identifier.otherb49337397en_US
dc.identifier.other51973306en_US
dc.identifier.otherThesis 51777en_US
dc.identifier.urihttp://hdl.handle.net/1773/9223
dc.descriptionThesis (Ph. D.)--University of Washington, 2002en_US
dc.description.abstractEndonucleases are a large class of enzymes that catalyze the formation of site-specific double strand breaks in DNA. Homing endonucleases are expressed from open reading frames within intervening sequences (introns or inteins) and promote the lateral transfer of their host intron to a cognate intronless allele. A novel mechanism of DNA endonucleolytic cleavage, exhibited by the homing endonuclease I-Ppol and characterized by a histidine general base, has been visualized at high resolution by intermediate trapping and studied computationally. The structures of three separate catalytic species were determined by X-ray crystallography and are presented here. The nudged elastic band method was used to calculate the minimum energy path for the catalyzed reaction and the predicted rate is compared to experiment. In addition, the structure of I-Ppol bound to its DNA target site shows that bound DNA is severely bent, resulting in significant deformations of the minor and major grooves near the scissile phosphates. To study the role of conformational changes within the protein catalyst and the DNA substrate, the structures of both the apo enzyme and the L116A mutant protein/DNA complex with severely decreased binding affinity were determined. The wild-type protein displays a rigid-body rotation of its individual subunits upon DNA binding. Homing site DNA is not detectably bent in the absence of protein, but is sharply bent in both the WT and L116A complexes, indicating that binding involves a large distortion of the DNA and a smaller change in protein conformation. The critical function of leucine 116 is also described.T4 phage polynucleotide kinase (PNK) was identified over 35 years ago and has become a staple reagent for molecular biologists. The enzyme displays 5'-hydroxyl kinase, 3'-phosphatase, and 2',3'-cyclic phosphodiesterase activities against a wide range of substrates. These activities modify the ends of a nicked tRNA generated by a bacterial response to infection and facilitate their repair by T4 RNA ligase. DNA repair enzymes that share conserved motifs with PNK have been identified in eukaryotes. PNK contains two functionally distinct structural domains and forms a homotetramer. The C-terminal phosphatase domain is homologous to the L-2 haloacid dehalogenase family and the N-terminal kinase domain is homologous to adenylate kinase. The active sites have been characterized through structural homology analyses and visualization of bound substrate.en_US
dc.format.extentxi, 162 p.en_US
dc.language.isoen_USen_US
dc.rightsCopyright is held by the individual authors.en_US
dc.rights.urien_US
dc.subject.otherTheses--Biological chemistryen_US
dc.titleStructural and computational studies of two oligonucleotide modifying enzymes: I-PpoI and T4 polynucleotide kinaseen_US
dc.typeThesisen_US


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