Lysogeny is a defining feature of temperate bacteriophages. Temperate bacteriophages are able to establish lysogeny by integrating into the host chromosome or extrachromosomally, as a plasmid-like prophage in the host cytoplasm. In either case, host factors are involved in both the establishment and maintenance of lysogeny. Mycobacteriophage L5 forms an integrated prophage through the action of a phage-encoded tyrosine recombinase. L5 integrase (Int) binds to the phage attachment site, attP, and the bacterial attachment site, attB. Int binds attP bivalently by binding to the core, where strand exchange occurs, and to arm-type binding sites that flank the core. A host-encoded DNA binding protein, mIHF, is required for recombination and binds between the core and arm-type binding sites of attP. mIHF is thought to catalyze the bending of attP required for bivalent binding between the core and arm-type binding sites. attP core consists of a seven base pair overlap region flanked on either side by imperfect inverted repeats that make up the recombinase binding elements or RBEs. The RBEs were shown to be essential for core binding by creating attPs with mutations in the RBEs. When both of the RBEs are mutated Int can neither perform recombination, nor create specific complexes that involve core binding. When the right side RBE is wild type with mutant left side RBE, recombination can occur, and complexes involving core binding are observed. However, Int is unable to catalyze recombination when the left side RBE is wild type and the right is mutated, indicating that contact must be made with attP on the right side of core for a stable Int/attP core complex to be formed. The middle domain connects the two outer domains, but its function in recombination is not well understood. To determine the function of the middle domain, mutations were made at conserved residues in the middle domain of Int, and these mutants were characterized. These mutants are able to catalyze recombination, but do not form the recombinagenic complexes observed by wild type Int. Increasing the mIHF concentration in these reactions augments the recombination efficiency and stabilizes recombinagenic complexes that involve Int/core binding.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12162002-131450 |
| Date | 12 March 2003 |
| Creators | Wadsworth, Curtis Carl |
| Contributors | Graham F. Hatfull, Roger W. Hendrix, James Pipas, Linda Jen-Jacobson, Saleem Khan |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu:80/ETD/available/etd-12162002-131450/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0018 seconds