Functional characterization of a phage integrase and its possible use in gene therapy
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Bacteriophage P2 infecting Escherichia coli can integrate into the bacterial chromosome by site-specific recombination, which is catalyzed by the P2 Int recombinase. The recombination event takes place between the phage attachment site, attP, and the bacterial attachment site, attB. Once integrated into the host chromosome the P2 prophage is very stable since an additional phage protein, Cox, is required for excision. For both integration and excision, the host-encoded protein IHF is also required.
In this thesis, I have made a functional characterization of the P2 integrase and investigated its future potential as a tool for gene therapy. The P2 integrase was found to have cooperative interactions upon DNA binding with its accessory proteins, IHF and Cox. An N-terminal truncated Int protein retained these cooperative interactions, although it was disrupted in arm-binding. Moreover, the Int protein was found to form stable dimers in the absence of DNA, and the C-terminus and amino acid E197 was found to be important for dimerization. Dimerization was found to be essential for recombination, but dimerization deficient mutant proteins were able to bind as well as the wt protein to attP.
The P2 Int was found to mediate recombination with a human sequence at a low frequency. It was also found that the insertion of HMG-recognition boxes can substitute for the requirement of IHF for recombination in an eukaryotic cell extract and that the integrase protein is localized in the cell nucleus. Taken together, these results indicate that the P2 integrase could be of potential use in gene therapy.
Place, publisher, year, edition, pages
Stockholm: Institutionen för genetik, mikrobiologi och toxikologi , 2005. , 49 p.
IdentifiersURN: urn:nbn:se:su:diva-397ISBN: 91-7155-015-1OAI: oai:DiVA.org:su-397DiVA: diva2:193375
2005-03-18, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 13:00 (English)
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