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A comparative analysis of the bifunctional Cox proteins of two heteroimmune P2-like phages with different host integration sites
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)
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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.

Place, publisher, year, edition, pages
2009. Vol. 385, no 2, 303-12 p.
Keyword [en]
Bacteriophage, Directionality factor, Repressor
Identifiers
URN: urn:nbn:se:su:diva-31971DOI: 10.1016/j.virol.2008.12.002ISI: 000264252200003PubMedID: 19150106OAI: oai:DiVA.org:su-31971DiVA: diva2:279079
Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2017-12-12Bibliographically 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: 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
3. 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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