A phage protein that binds φC31 integrase to switch its directionality

The serine integrase, Int, from the Streptomyces phage φC31 mediates the integration and excision of the phage genome into and out of the host chromosome. Integrases usually require a recombination directionality factor (RDF) or Xis to control integration and excision and, as φC31 Int only mediates integration in the absence of other phage proteins, we sought to identify a φC31 RDF. Here we report that the φC31 early protein, gp3 activated attL x attR recombination and inhibited attP x attB recombination. Gp3 binds to Int in solution and when Int is bound to the attachment sites. Kinetic analysis of the excision reaction suggested that gp3 modifies the interactions between Int and the substrates to form an active recombinase. In the presence of gp3, Int assembles an excision synaptic complex and the accumulation of the integration complex is inhibited. The structure of the excision synaptic complex, like that of the hyperactive mutant of Int, IntE449K, appeared to be biased towards one that favours the production of correctly joined products. The functional properties of φC31 gp3 resemble those of the evolutionarily unrelated RDF from phage Bxb1, suggesting that these two RDFs have arisen through convergent evolution.

[1]  C. A. Thomas,et al.  Molecular cloning. , 1977, Advances in pathobiology.

[2]  L. Mellaert,et al.  Complete genomic nucleotide sequence and analysis of the temperate bacteriophage VWB. , 2005, Virology.

[3]  P. Yuan,et al.  Tetrameric structure of a serine integrase catalytic domain. , 2008, Structure.

[4]  D. Esposito,et al.  Identification of an HP1 phage protein required for site‐specific excision , 1994, Molecular microbiology.

[5]  R. Pérez-Redondo,et al.  Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces , 2005, Nucleic acids research.

[6]  E. Haggård-Ljungquist,et al.  Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox. , 2005, Virology.

[7]  Andrew R McEwan,et al.  Site-specific recombination by phiC31 integrase and other large serine recombinases. , 2010, Biochemical Society transactions.

[8]  Paul A. Rowley,et al.  A motif in the C-terminal domain of ϕC31 integrase controls the directionality of recombination , 2008, Nucleic acids research.

[9]  W. Brown,et al.  Iterative in vivo assembly of large and complex transgenes by combining the activities of φC31 integrase and Cre recombinase , 2005, Nucleic acids research.

[10]  Paul A. Rowley,et al.  DNA binding and synapsis by the large C-terminal domain of ϕC31 integrase , 2009, Nucleic acids research.

[11]  Margaret C. M. Smith,et al.  Switching the polarity of a bacteriophage integration system , 2004, Molecular microbiology.

[12]  L. Mellaert,et al.  Site-specific integration of bacteriophage VWB genome into Streptomyces venezuelae and construction of a VWB-based integrative vector. , 1998, Microbiology.

[13]  A. Landy,et al.  λ Integrase and the λ Int Family , 2002 .

[14]  Hugo J. Bellen,et al.  P[acman]: A BAC Transgenic Platform for Targeted Insertion of Large DNA Fragments in D. melanogaster , 2006, Science.

[15]  J. Rood,et al.  The large resolvase TnpX is the only transposon‐encoded protein required for transposition of the Tn4451/3 family of integrative mobilizable elements , 2004, Molecular microbiology.

[16]  N. Grindley,et al.  Mechanisms of site-specific recombination. , 2003, Annual review of biochemistry.

[17]  M. Calos.  The phiC31 integrase system for gene therapy. , 2006, Current gene therapy.

[18]  P. Mullany,et al.  The Large Resolvase TndX Is Required and Sufficient for Integration and Excision of Derivatives of the Novel Conjugative Transposon Tn5397 , 2000, Journal of bacteriology.

[19]  J. Lewis,et al.  Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins. , 2001, Nucleic acids research.

[20]  M. Gregory,et al.  The complete genome sequence of the Streptomyces temperate phage φC31: evolutionary relationships to other viruses , 1999 .

[21]  D. Ladant,et al.  A bacterial two-hybrid system based on a reconstituted signal transduction pathway. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Margaret C. M. Smith,et al.  Synapsis and DNA cleavage in phiC31 integrase-mediated site-specific recombination. , 2004, Nucleic acids research.

[23]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[24]  K. Hammer,et al.  Novel Organization of Genes Involved in Prophage Excision Identified in the Temperate Lactococcal Bacteriophage TP901-1 , 1999, Journal of bacteriology.

[25]  E. Haggård-Ljungquist,et al.  The Cox protein is a modulator of directionality in bacteriophage P2 site-specific recombination , 1993, Journal of bacteriology.

[26]  P. Ritzenthaler,et al.  Control of Directionality in Bacteriophage mv4 Site-Specific Recombination: Functional Analysis of the Xis Factor , 2009, Journal of bacteriology.

[27]  Margaret C. M. Smith,et al.  Control of directionality in the site‐specific recombination system of the Streptomyces phage φC31 , 2000, Molecular microbiology.

[28]  DNA binding and synapsis by the large C-terminal domain of r C 31 integrase , 2009 .

[29]  J. Naismith,et al.  A simple and efficient expression and purification system using two newly constructed vectors. , 2009, Protein expression and purification.

[30]  M. Gregory,et al.  Integration Site for Streptomyces Phage φBT1 and Development of Site-Specific Integrating Vectors , 2003, Journal of bacteriology.

[31]  Shruti Jain,et al.  Mycobacteriophage Bxb 1 integrates into the Mycobacterium smegmatis groEL 1 gene , 2003 .

[32]  Graham F Hatfull,et al.  Integration and excision by the large serine recombinase φRv1 integrase , 2005, Molecular microbiology.

[33]  G. Hatfull,et al.  Two-step site selection for serine-integrase-mediated excision: DNA-directed integrase conformation and central dinucleotide proofreading , 2008, Proceedings of the National Academy of Sciences.

[34]  Margaret C. M. Smith,et al.  Diversity in the serine recombinases , 2002, Molecular microbiology.

[35]  J. Brookfield,et al.  Rearranging the centromere of the human Y chromosome with φC31 integrase , 2005, Nucleic acids research.

[36]  Xiaoling Zhang,et al.  Complete genomic sequence analysis of the temperate bacteriophage phiSASD1 of Streptomyces avermitilis. , 2010, Virology.

[37]  Shruti Jain,et al.  Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene , 2003, Molecular microbiology.

[38]  Margaret C. M. Smith,et al.  In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[39]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[40]  Graham F Hatfull,et al.  Synapsis in phage Bxb1 integration: selection mechanism for the correct pair of recombination sites. , 2005, Journal of molecular biology.

[41]  G. Hatfull,et al.  The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB. , 2003, Molecular cell.

[42]  R. Clubb,et al.  Identification of the λ integrase surface that interacts with Xis reveals a residue that is also critical for Int dimer formation , 2003, Proceedings of the National Academy of Sciences of the United States of America.