Mutational analysis of integrase and excisionase proteins in bacteriophage lambda site-specific recombination system
Wu, Zhao
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https://hdl.handle.net/2142/18938
Description
Title
Mutational analysis of integrase and excisionase proteins in bacteriophage lambda site-specific recombination system
Author(s)
Wu, Zhao
Issue Date
1995
Doctoral Committee Chair(s)
Gardner, Jeffrey F.
Committee Member(s)
Gumport, Richard I.
Voss, Edward W., Jr.
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, Molecular
Biology, Microbiology
Language
eng
Abstract
Second-site reversion analyses were performed on 21 recombination defective Int mutants. A second-site revertant P243L/E218K and a pseudorevertant E175K were isolated and further characterized using genetic and biochemical methods. The results showed that the second-site substitution E218K selectively enhances Int core-binding affinity and acts as a global suppressor. In addition, our genetic data suggest that position 218 is part of or near the putative Int core-binding domain.
Saturation mutagenesis was performed on the region of the xis gene that encodes carboxyl region of the Xis protein. Forty-five new C-terminal region mutants were isolated and their abilities to promote excision, to bind the DNA and to interact with Int and FIS were characterized in vivo and in vitro. In addition, the secondary structure of this region was predicted by computer modeling schemes. A model for the secondary structures of carboxyl region of Xis protein was developed by combining the mutagenesis information with computer-based analytic techniques. According to the model, residues between 57 to 65 are located in an $\alpha$-helix structure. The functional study of mutants within the helix suggests that one side of the helix (residues 57, 60, 63 and 64) interacts with Int. In addition, the identification of structurally stable mutants of Xis may be useful in studying its degradation pathway and in supplying protein for crystallographic studies.
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