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Functional characterization of a phage integrase and its possible use in gene therapy
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:su:diva-397ISBN: 91-7155-015-1 (print)OAI: oai:DiVA.org:su-397DiVA: diva2:193375
Public defence
2005-03-18, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2005-02-24 Created: 2005-02-24 Last updated: 2010-05-05Bibliographically approved
List of papers
1. Dimerization of bacteriophage P2 integrase is not required for binding to its DNA target but for its biological activity
Open this publication in new window or tab >>Dimerization of bacteriophage P2 integrase is not required for binding to its DNA target but for its biological activity
2005 In: Gene, ISSN 0378-1119, Vol. 344, 221-231 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23609 (URN)
Note
Part of urn:nbn:se:su:diva-397Available from: 2005-02-24 Created: 2005-02-24Bibliographically approved
2. Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox
Open this publication in new window or tab >>Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox
2005 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 332, no 1, 284-294 p.Article in journal (Refereed) Published
Abstract [en]

Bacteriophage P2 integrase (Int) mediates site-specific recombination leading to integration or excision of the phage genome in or out of the bacterial chromosome. Int belongs to the large family of tyrosine recombinases that have two different DNA recognition motifs binding to the arm and core sites, respectively, which are located within the phage attachment sites (attP). In addition to the P2 integrase, the accessory proteins Escherichia coli IHF and P2 Cox are needed for recombination. IHF is a structural protein needed for integration and excision by bending the DNA. As opposed to lambda, only one IHF site is found in P2 attP. P2 Cox controls the direction of recombination by inhibiting integration but being required for excision. In this work, the effects of accessory proteins on the capacity of Int to bind to its DNA recognition sequences are analyzed using electromobility shifts. P2 Int binds with low affinity to the arm site, and this binding is greatly enhanced by IHF. The arm binding domain of Int is located at the N-terminus. P2 Int binds with high affinity to the core site, and this binding is also enhanced by IHF. The fact that the cooperative binding of Int and IHF is strongly reduced by lengthening the distance between the IHF and core binding sites indicates that the distance between these sites may be important for cooperative binding. The Int and Cox proteins also bind cooperatively to attP.

Keyword
Bacteriophage, Site-specific recombination, Integrase, IHF, Cox, Directionality factor
Identifiers
urn:nbn:se:su:diva-23610 (URN)10.1016/j.virol.2004.11.015 (DOI)
Available from: 2005-02-24 Created: 2005-02-24 Last updated: 2010-05-12Bibliographically approved
3. Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells
Open this publication in new window or tab >>Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells
Show others...
2008 (English)In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 105, no 1, 290-299 p.Article in journal (Refereed) Published
Abstract [en]

AIMS: To investigate if the site-specific tyrosine integrase (Int) from phage P2 has features that would make it interesting for use of gene transfer into eukaryotic cells. These include the possibility of promoting recombination with a nonphage sequence, abolishing the requirement for the bacterial DNA-binding and -bending protein integration host factor (IHF), and localization to the nucleus of eukaryotic cells. METHODS AND RESULTS: We show that the Int protein catalyzes site-specific recombination using a human sequence in Escherichia coli and in vitro although not as efficiently as with the wild-type bacterial sequence, and that insertion of high mobility group recognition boxes in the phage attachment site substrate abolish the requirement of IHF and allows efficient recombination in vitro in a eukaryotic cell extract. Furthermore, we show by fluorescence that the Int protein contains a functional intrinsic nuclear localization signal, localizing it to the nucleus in both HeLa and 293 cells. CONCLUSIONS: We conclude that P2 Int may be a potential tool for site-specific integration of genes into the human chromosome. SIGNIFICANCE AND IMPACT OF THE STUDY: The study implies the possibility of using multiple prokaryotic Int proteins with different specific integration sites in human cells for future gene therapy programmes.

Keyword
bacteriophage, integrase, nuclear localization signal, site-specific recombination
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-38940 (URN)10.1111/j.1365-2672.2008.03748.x (DOI)000256494600032 ()
Available from: 2010-05-04 Created: 2010-05-04 Last updated: 2010-05-12Bibliographically approved

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