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https://hdl.handle.net/2142/72514
Description
Title
Characterization of the Gifsy-1 Phage Excisionase
Author(s)
Flanigan, Asa
Issue Date
2008
Doctoral Committee Chair(s)
Gardner, Jeffrey F.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
The Gifsy-1 phage is a temperate bacterial virus that infects its host, Salmonella enterica serovar Typhimurium. During lysogeny, the phage integrates site-specifically into a single bacterial attachment sequence in the Salmonella chromosome. The reaction involves recombination between the phage attachment sequence (attP) and the bacterial attachment sequence (attB). The phage encoded enzyme, Gifsy-1 integrase (Int), catalyzes this recombination reaction between the phage chromosome and the bacterial host chromosome. The reaction requires and accessory protein, Integration Host Factor (IHF). This forward recombination reaction between phage encoded attP and host encoded attB is referred to as phage integration. Integration results in the creation of two attachment sites that border the integrated phage chromosome, attL and attR. It is believed that the Gifsy-1 phage Int catalyzes a reaction that is chemically identical to that described for the lambda Int protein. This reaction results in chimeric products of the recombination reaction, attL and attR, in which one-half of the att sequence is composed of host DNA and the remainder is composed of phage DNA. The reaction is not directly reversible and requires another phage encoded protein, the Gifsy-1 excisionase (Xis) protein for the integrated phage to excise from the host chromosome.
This study focuses on the structure and function Gifsy-1 Xis protein. I have characterized the specific binding sites of the Gifsy-1 Xis protein. My methods include well characterized EMSA and DNA footprinting techniques. Our quantitative results have allowed us to propose a model that describes the binding by which protomers of the Gifsy-1 Xis protein interact with attP DNA. In addition, I have begun to study the structure of the Xis protein through mutational analysis, multiple sequence analysis, 3-dimensional de novo modeling, and limited proteolysis. I have assigned potential functional motifs to fragments of the Gifsy-1 Xis protein through modeling and sequence analysis. My results were substantiated through mutational analysis and limited proteolysis of the wild-type Gifsy-1 Xis protein and Gifsy-1 Xis substitution mutants.
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