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Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Elisabeth Haggård-Ljungquist)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Elisabeth Haggård-Ljungquist)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday)
Department of Clinical Chemistry, Danderyd Hospital and Karolinska Institute, Stockholm, Sweden.
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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.

Place, publisher, year, edition, pages
2008. Vol. 105, no 1, 290-299 p.
Keyword [en]
bacteriophage, integrase, nuclear localization signal, site-specific recombination
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-38940DOI: 10.1111/j.1365-2672.2008.03748.xISI: 000256494600032OAI: oai:DiVA.org:su-38940DiVA: diva2:317641
Available from: 2010-05-04 Created: 2010-05-04 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Functional characterization of a phage integrase and its possible use in gene therapy
Open this publication in new window or tab >>Functional characterization of a phage integrase and its possible use in gene therapy
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:nbn:se:su:diva-397 (URN)91-7155-015-1 (ISBN)
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
2. Site-Specific Recombination: Integrases, Accessory Factors and DNA Targets of P2-like Coliphages
Open this publication in new window or tab >>Site-Specific Recombination: Integrases, Accessory Factors and DNA Targets of P2-like Coliphages
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The temperate coliphage P2 and its family members integrate their genomes into the host Escherichia coli chromosome by a site-specific recombination mechanism to form lysogeny. Integration takes place between the complex phage attP site and the simple bacterial attB site and is catalyzed by the phage encoded integrase (Int). Similar to the archetype λ Int, the P2-like phage integrases are heterobivalent tyrosine recombinases which possess the ability to simultaneously bind two different and distant types of DNA sequences within the attP region. To bridge the core and the flanking arm-binding sites in attP, the integrase requires the assistance of accessory factors that bend the DNA; the host encoded IHF and the phage encoded Cox protein. Cox acts as a directionality factor by being required for integration but is inhibitory for the excisive reaction.

The purpose of this doctoral thesis has been to gain a more detailed knowledge of the site-specific recombination systems of phages P2 and WΦ, which are close relatives but integrate into different host targets. The future aim is to develop these systems for targeted integration into the genome of higher eukaryotes.

The P2 Int and an N-terminal truncation of the integrase were shown to bind cooperatively together with IHF or Cox to the DNA targets, however the N-truncated protein lost its ability to bind to the arm sequence. WΦ Cox was shown to bind cooperatively with WΦ Int to attP whereas the opposite was evident for WΦ Cox and IHF. The 27 nucleotides that are identical between the core and attB of phage P2 were investigated for their importance in binding and recombination. The right part of the core was shown to be the primary Int binding site where one single base substitution was shown to abolish P2 Int binding and recombination. An alanine scanning of the two predicted alpha-helices in the presumed core-binding domain of P2 Int was carried out in order to identify amino acids involved in binding to the core. An in vivo excisive assay and an in vivo integrative assay were used resulting in the identification of four amino acids as candidates for core-binding. The fact that the recombination reaction shows directionality renders the site-specific recombination systems of the P2-like phages attractive to develop as tools for safe and efficient non-viral gene delivery in humans. The wild-type P2 integrase was shown to accept a human attB sequence and localizes to the nucleus in human cell lines.

The work presented in this thesis has increased our understanding of the site-specific recombination systems of the phages P2 and WΦ and provides a basis for further characterization and development for future use in a eukaryotic context.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2010. 51 p.
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-38914 (URN)978-91-7447-092-5 pp. 1-51 (ISBN)
Public defence
2010-06-04, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.Available from: 2010-05-11 Created: 2010-05-04 Last updated: 2010-05-12Bibliographically approved

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