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Identification of bases required for P2 Integrase core-binding and recombination
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Elisabeth Haggård-Ljungquist)
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. (Elisabeth Haggård-Ljungquist)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Elisabeth Haggård-Ljungquist)
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Temperate coliphage P2 integrates its genome into the host chromosome upon lysogenization via a site-specific recombination event mediated by an integrase belonging to the complex family of tyrosine recombinases. The host integration site attB (BOB´) is localized in the end of the cyaR gene and share 27 nucleotides with the core of attP (COC´). In the present study we determine the minimal attB site using an in vivo recombination assay and 10 nt on the left side (B) are found to be nonessential for recombination. We show that the integrase has higher affinity for the right side (B´) compared to B and that artificial B´OB´ and an attP site with a matching core (C´OC´) are efficient substrates for recombination in vitro. We have analyzed single nucleotides in attB and find that sequence homology within a non-centrally located quadruplet in the hypotetical overlap region is essential for efficient recombination in vivo.

Keyword [en]
Site-specific recombination, integrase, tyrosine recombinase, bacteriophage
National Category
Genetics Cell and Molecular Biology Microbiology in the medical area
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-38925OAI: oai:DiVA.org:su-38925DiVA: diva2:317568
Available from: 2010-05-04 Created: 2010-05-04 Last updated: 2010-05-12Bibliographically approved
In thesis
1. 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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