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Site-specific recombination of P2-like phages; possible tools for safe gene therapy: A focus on phage ΦD145
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
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 [en]
bacteriophage, integrase, site-specific recombination
National Category
Natural Sciences
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-45940ISBN: 978-91-7447-174-8 (print)OAI: oai:DiVA.org:su-45940DiVA: diva2:370488
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
List of papers
1. Characterization of the site-specific recombination system of phage ΦD145, and its capacity to promote recombination in human cells
Open this publication in new window or tab >>Characterization of the site-specific recombination system of phage ΦD145, and its capacity to promote recombination in human cells
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.

Keyword
Integrase, Site-specific recombination, Bacteriophage
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-25724 (URN)10.1016/j.virol.2010.08.035 (DOI)000283970500007 ()
Available from: 2009-02-12 Created: 2009-02-03 Last updated: 2017-12-13Bibliographically approved
2. Phosphorylation affects the biological activity of the integrase of the P2-likephage ΦD145
Open this publication in new window or tab >>Phosphorylation affects the biological activity of the integrase of the P2-likephage ΦD145
(English)Manuscript (preprint) (Other academic)
Keyword
Integrase, Phage ΦD145; Protein phosphorylation; Wzc
Identifiers
urn:nbn:se:su:diva-45939 (URN)
Available from: 2010-11-16 Created: 2010-11-16 Last updated: 2010-11-17Bibliographically approved
3. A structure-function analysis of P2 integrase
Open this publication in new window or tab >>A structure-function analysis of P2 integrase
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Bacteriophage P2 integrase catalyzes site-specific recombination between the phage DNA and the host chromosome thereby promoting integration or excision of the phage genome. P2 integrase belongs to the large tyrosine family of integrases that shows little sequence identity besides some conserved boxes and patches in the catalytic domain. However, the overall structure of the tyrosine family of integrases seems to be similar. Phage integrases have the potential as tools for site-specific gene insertions into eukaryotic genomes provided that target sequences are available. To elucidate the possibility of evolving the P2 integrase to accept new targets, we have in this work initiated a structure-function analysis of the P2 integrase using two approaches based on a comparison of the predicted secondary structure of P2 integrase with that determined for the lambda integrase. First, we have made hybrids between P2 integrase and the related WΦ integrase that has a different host DNA target, to locate the region promoting specificity between the integrases. This, however, has not been possible, the N-terminal domains can be exchanged without losing biological activity and this will not affect the specificity. All other hybrids made were biological inactive. Next we have made an alanine scanning of the alpha helices believed to be involved in specific interactions with the target, and four amino acids have been identified as candidates for sequence-specific interactions with the core.

Keyword
site-specific recombination, integrase, tyrosine recombinase, bacteriophage
National Category
Genetics Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-38927 (URN)
Note
Authors 1 and 2: Equal contribution to this workAvailable from: 2010-05-04 Created: 2010-05-04 Last updated: 2010-11-17Bibliographically approved
4. A comparative analysis of the bifunctional Cox proteins of two heteroimmune P2-like phages with different host integration sites
Open this publication in new window or tab >>A comparative analysis of the bifunctional Cox proteins of two heteroimmune P2-like phages with different host integration sites
Show others...
2009 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 385, no 2, 303-12 p.Article in journal (Refereed) Published
Abstract [en]

The Cox protein of the coliphage P2 is multifunctional; it acts as a transcriptional repressor of the Pc promoter, as a transcriptional activator of the P(LL) promoter of satellite phage P4, and as a directionality factor for site-specific recombination. The Cox proteins constitute a unique group of directionality factors since they couple the developmental switch with the integration or excision of the phage genome. In this work, the DNA binding characteristics of the Cox protein of WPhi, a P2-related phage, are compared with those of P2 Cox. P2 Cox has been shown to recognize a 9 bp sequence, repeated at least 6 times in different targets. In contrast to P2 Cox, WPhi Cox binds with a strong affinity to the early control region that contains an imperfect direct repeat of 12 nucleotides. The removal of one of the repeats has drastic effects on the capacity of WPhi to bind to the Pe-Pc region. Again in contrast to P2 Cox, WPhi Cox has a lower affinity to attP compared to the Pe-Pc region, and a repeat of 9 bp can be found that has 5 bp in common with the repeat in the Pe-Pc region. WPhi Cox, however, is essential for excisive recombination in vitro. WPhi Cox, like P2 Cox, binds cooperatively with integrase to attP. Both Cox proteins induce a strong bend in their DNA targets upon binding.

Keyword
Bacteriophage, Directionality factor, Repressor
Identifiers
urn:nbn:se:su:diva-31971 (URN)10.1016/j.virol.2008.12.002 (DOI)000264252200003 ()19150106 (PubMedID)
Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2017-12-12Bibliographically approved

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