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|Title:||Genetic and mechanistic studies of the bacteriophage lambda integrase protein|
|Author(s):||Han, Yiping Weng|
|Doctoral Committee Chair(s):||Gardner, Jeffrey F.|
|Department / Program:||Microbiology|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Site-specific recombination between bacteriophage $\lambda$ and E. coli chromosomes starts with the formation of higher-order nucleoprotein complexes, called "intasomes", followed by a series of steps catalyzed by the phage-encoded integrase (Int). In order to study the functions of Int during recombination, Int mutants defective in in vivo excision were isolated and screened for proficiency in binding to the arm-type sites using challenge phage assays. Fifty-eight such mutants were obtained and were further characterized (1) for their ability to bind the P$\sp\prime$1 and P$\sp\prime$123 arm-type sites and to from attL intasomes, (2) for negative dominance in vitro, (3) for the type I topoisomerase activity, and (4) for the ability to resolve recombination intermediates. I found that (1) residues in a stretch of 88 amino acids in the middle of the protein may be involved in Int-Int interactions, (2) a region around Arg212 is part of the active site, (3) residues in the carboxyl terminal play a role in enhancing Int binding to its arm-type sites, possibly by interacting with the small amino terminal region that is responsible for specific recognition of the arm-type sites, and (4) residues at the very carboxyl end of the protein may be involved in modulating the cleavage or religation activities of the protein.
Some of the Int mutants were then used to study the recombination mechanism. I performed in vitro complementation assays for strand cleavage using attL suicide substrates and mutant proteins containing amino acid substitutions at residues conserved in the integrase family of recombinases. I demonstrated that at least two Int monomers are required to form the active site that performs cleavage at the B site in an attL complex. The Tyr342 residue that contains the nucleophile and cleaves the B site is provided by an Int monomer that is not itself bound to the B site, i.e., cleavage is by a trans mechanism. The cleavage of the B site is intra-molecular. In addition, I proposed a model in which cleavage and synapsis are coupled during excisive recombination.
|Rights Information:||Copyright 1993 Han, Yiping Weng|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9329051|