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|Title:||A genetic analysis of bacteriophage lambda integrase-core site interactions|
|Author(s):||MacWilliams, Maria Patrice|
|Doctoral Committee Chair(s):||Gardner, Jeffrey F.|
|Department / Program:||Microbiology|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|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.
|Rights Information:||Copyright 1992 MacWilliams, Maria Patrice|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9236529|