Genetic and mechanistic studies of the bacteriophage lambda integrase protein
Han, Yiping Weng
This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.
Permalink
https://hdl.handle.net/2142/19633
Description
Title
Genetic and mechanistic studies of the bacteriophage lambda integrase protein
Author(s)
Han, Yiping Weng
Issue Date
1993
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, Genetics
Biology, Microbiology
Language
eng
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.
Use this login method if you
don't
have an
@illinois.edu
email address.
(Oops, I do have one)
IDEALS migrated to a new platform on June 23, 2022. If you created
your account prior to this date, you will have to reset your password
using the forgot-password link below.
