University of Illinois Urbana-Champaign

A genetic analysis of bacteriophage lambda integrase-core site interactions

MacWilliams, Maria Patrice

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https://hdl.handle.net/2142/23413

Description

Title
A genetic analysis of bacteriophage lambda integrase-core site interactions
Author(s)
MacWilliams, Maria Patrice
Issue Date
1992
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, Molecular
  • Biology, Genetics
  • Biology, Microbiology
Language
eng
Abstract
  • Bacteriophage lambda utilizes a site-specific recombination system to integrate its DNA into the E. coli chromosome during lysogeny. The viral protein, Integrase (Int), catalyzes strand exchange between the phage attachment site, attP, and the host target sequence, attB to generate the prophage sites, attL and attR. attP contains the core region, where strand exchange occurs, and the required flanking sequences, the P and P$\sp\prime$ arms. Within the core, there are two Int core-type binding sites. The flanking arm sequences contain binding sites for Int as well as for IHF, Xis, and FIS. This array of protein-DNA interactions forms a higher-order complex, the intasome, which is the active substrate for recombination.
  • Using the P22-based challenge-phage system, we have developed an in vivo assay to analyze both core binding and the long range interactions required for attL complex formation. The DNA sequences for the core alone as well as for the core plus the P$\sp\prime$ arm of attP have been cloned into the operator region and thus regulate the P22 antirepressor (ant) operon. In this system, the core sequence alone is not sufficient repression. However, in the attL (I) challenge phage, the P$\sp\prime$ arm enhances core-Int interaction so that transcription is inhibited. In addition, the core and H$\sp\prime$ sites enhance Int-arm type binding as well. These long range interactions require both Int and IHF and suggest that IHF, interacting with its H$\sp\prime$ site, bends the DNA so that the Int molecules bound to the arm-type sites are brought into close proximity to and thus can interact with the core. By a combination mutagenesis techniques, we have shown that the major binding-site determinants of the attL complex are the C$\sp\prime,$ H$\sp\prime,$ and P$\sp\prime$1 sites. Both the C and the P$\sp\prime$2 sites are also involved in complex formation.
  • Finally, I have used these attL challenge phage to characterize the DNA binding abilities of a group of Int mutants. With this analysis, I have been able to show that the evolutionarily conserved (his)-308 and (arg)-311 residues play a role in core binding.
Type of Resource
text
Permalink
http://hdl.handle.net/2142/23413
Copyright and License Information
Copyright 1992 MacWilliams, Maria Patrice

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Graduate Dissertations and Theses at Illinois PRIMARY
Graduate Theses and Dissertations at Illinois
Dissertations and Theses - Microbiology