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Characterization of the site-specific recombination system of phage ΦD145, and its capacity to promote recombination in human cells
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
2010 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 408, no 1, 64-70 p.Article in journal (Refereed) Published
Abstract [en]

Phage integrases have the potential of becoming tools for safe site-specific integration of genes into unmodified human genomes. The P2-like phages have been found to have different bacterial host integration sites and consequently they have related integrases with different sequence specificities. In this work the site-specific recombination system of the P2-like phage ΦD145 is characterized. The minimal attB site is determined to 22 nt with 18 nt identity to the core region of attP. A non-coding sequence on the human chromosome 13 is shown to be a rather good substrate for recombination in vivo in bacteria as well as in a plasmid system in HeLa cells when HMG protein recognition sequences are inserted between the left arm-binding site and the core in the complex phage attachment site attP. Thus ΦD145 integrase that belongs to the tyrosine family shows potential as a tool for site-specific integration into the human genome.

Place, publisher, year, edition, pages
2010. Vol. 408, no 1, 64-70 p.
Keyword [en]
Integrase, Site-specific recombination, Bacteriophage
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:su:diva-25724DOI: 10.1016/j.virol.2010.08.035ISI: 000283970500007OAI: oai:DiVA.org:su-25724DiVA: diva2:200376
Available from: 2009-02-12 Created: 2009-02-03 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Site-specific recombination in P2-like coliphages
Open this publication in new window or tab >>Site-specific recombination in P2-like coliphages
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The scope of these studies has been to investigate the site-specific recombination systems of P2-like coliphages, both in an evolutionary perspective by a comparative analysis of related phages as well as in a functional perspective.

Surveys of P2-like phages in Escherichia coli isolated from nature reveal the existence of seven discrete immunity classes and three integration sites, one of them previously unknown. Phylogenetic analysis of the repressor proteins and other analyses show that homologous recombination plays a role in the appearance of new immunities. Other studies of P2-like prophages from sequenced genomes from public databases show that the P2-like phages grow in different γ-proteobacteria. Based on the type of immunity and site-specific recombination system they can be roughly subdivided in two distinct subgroups and some new host integration sites could be identified. Some of the host attachment sites have a high identity to the sequences in the human genome, making them interesting as potential tools for targeted gene insertions into unmodified human cells.

The functional studies have been focused on the identification of the determinants for site specificity, which is important for the use of the enzyme for targeted gene insertions into unmodified genomes. Two approaches have been used. In one, we have performed a structure-function analysis of P2 Int that has identified several presumptive residues involved in specific binding to the core sequence, all of them located in the same alpha-helix. This knowledge could be a base for an in vitro evolution of the integrase to enable it to accept new DNA targets with a high affinity. With respect to the excisionases from P2-like coliphages integrating in different sites, we found that they share some common features when they bind and bend to their DNA targets, but there are also significant differences, especially those related to the number of binding sites and the distribution of these and the IHF binding sites in the attP regions. In the other approach we have started to characterize the site-specific recombination system of another P2-like phage, ΦD145, that has a host target with a high identity to a site in the human genome. This looks promising since the human sequence can be used in vivo in E. coli with a rather high efficiency.

Place, publisher, year, edition, pages
Stockholm: Institutionen för genetik, mikrobiologi och toxikologi, 2009. 41 p.
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-8502 (URN)978-91-7155-817-6 (ISBN)
Public defence
2009-03-06, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2009-02-12 Created: 2009-02-03 Last updated: 2012-02-28Bibliographically approved
2. Site-specific recombination of P2-like phages; possible tools for safe gene therapy: A focus on phage ΦD145
Open this publication in new window or tab >>Site-specific recombination of P2-like phages; possible tools for safe gene therapy: A focus on phage ΦD145
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

P2-like bacteriophages integrate their genome into the E. coli host cell by a site-specific recombination event upon lysogenization. The integrative recombination occurs between a specific sequence in the phage genome, attP, and a specific sequence in the host genome, attB, generating the host-phage junctions attL and attR. The integration is mediated by the phage enzyme integrase (Int) and the host factor IHF. The excisive recombination takes place between attL and attR, and is mediated by Int, IHF and phage encoded protein Cox. For safe integration of foreign genes into eukaryotic chromosome a recombinases is necessary which can perform the integration site-specifically. P2-like phage integrases have the potential to become tools for safe gene therapy. Their target is simple but specific, and once integration has occurred it is very stable in the absence of the Cox protein. The site-specific recombination mechanism has to be understood at the molecular level. Therefore, I have initiated the characterization of the site-specific recombination system of the P2-like phage ΦD145. In this work, Int and IHF are shown to bind to the different attachment sites cooperatively. One of two possible inverted repeats in attP is shown to be the Int core recognition site. The attP core of this phage has high identity with a site on human chromosome, denoted as ΨattB. In this study we have shown that in in vivo recombination ΦD145 Int can accept ΨattB in both bacteria and in eukaryotic cells. Also shown that Int consists of an intrinsic nuclear localization signal. A study also reveled that ΦD145 Int activity was affected by the Tyr-phosphorylation. Attempts have been made to change the specificity of the other P2-like phage P2 and WΦ integrases and also structural and functional analysis was done. A study on comparative analysis of Cox proteins and Cox binding sites gave us the basic information about the recombination mechanism.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2010. 50 p.
Keyword
bacteriophage, integrase, site-specific recombination
National Category
Natural Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-45940 (URN)978-91-7447-174-8 (ISBN)
Public defence
2010-12-17, sal E306, 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 2: Manuscript. Paper 3: Manuscript.Available from: 2010-11-25 Created: 2010-11-16 Last updated: 2010-11-17Bibliographically approved

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