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Site-Specific Recombination: Integrases, Accessory Factors and DNA Targets of P2-like Coliphages
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Elisabeth Haggård-Ljungquist)
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: urn:nbn:se:su:diva-38914ISBN: 978-91-7447-092-5 pp. 1-51 OAI: oai:DiVA.org:su-38914DiVA: diva2:317560
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
List of papers
1. Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox
Open this publication in new window or tab >>Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox
2005 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 332, no 1, 284-294 p.Article in journal (Refereed) Published
Abstract [en]

Bacteriophage P2 integrase (Int) mediates site-specific recombination leading to integration or excision of the phage genome in or out of the bacterial chromosome. Int belongs to the large family of tyrosine recombinases that have two different DNA recognition motifs binding to the arm and core sites, respectively, which are located within the phage attachment sites (attP). In addition to the P2 integrase, the accessory proteins Escherichia coli IHF and P2 Cox are needed for recombination. IHF is a structural protein needed for integration and excision by bending the DNA. As opposed to lambda, only one IHF site is found in P2 attP. P2 Cox controls the direction of recombination by inhibiting integration but being required for excision. In this work, the effects of accessory proteins on the capacity of Int to bind to its DNA recognition sequences are analyzed using electromobility shifts. P2 Int binds with low affinity to the arm site, and this binding is greatly enhanced by IHF. The arm binding domain of Int is located at the N-terminus. P2 Int binds with high affinity to the core site, and this binding is also enhanced by IHF. The fact that the cooperative binding of Int and IHF is strongly reduced by lengthening the distance between the IHF and core binding sites indicates that the distance between these sites may be important for cooperative binding. The Int and Cox proteins also bind cooperatively to attP.

Keyword
Bacteriophage, Site-specific recombination, Integrase, IHF, Cox, Directionality factor
Identifiers
urn:nbn:se:su:diva-23610 (URN)10.1016/j.virol.2004.11.015 (DOI)
Available from: 2005-02-24 Created: 2005-02-24 Last updated: 2017-12-13Bibliographically approved
2. 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
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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.

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
3. Identification of bases required for P2 Integrase core-binding and recombination
Open this publication in new window or tab >>Identification of bases required for P2 Integrase core-binding and recombination
(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
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:nbn:se:su:diva-38925 (URN)
Available from: 2010-05-04 Created: 2010-05-04 Last updated: 2010-05-12Bibliographically approved
4. A structure-function analysis of P2 integrase
Open this publication in new window or tab >>A structure-function analysis of P2 integrase
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(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
5. Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells
Open this publication in new window or tab >>Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells
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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.

Keyword
bacteriophage, integrase, nuclear localization signal, site-specific recombination
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-38940 (URN)10.1111/j.1365-2672.2008.03748.x (DOI)000256494600032 ()
Available from: 2010-05-04 Created: 2010-05-04 Last updated: 2017-12-12Bibliographically approved

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