DNA cleavage is independent of synapsis during Streptomyces phage phiBT1 integrase-mediated site-specific recombination.

Bacteriophage-encoded serine recombinases have great potential in genetic engineering but their catalytic mechanisms have not been adequately studied. Integration of ϕBT1 and ϕC31 via their attachment (att) sites is catalyzed by integrases of the large serine recombinase subtype. Both ϕBT1 and ϕC31 integrases were found to cleave single-substrate att sites without synaptic complex formation, and ϕBT1 integrase relaxed supercoiled DNA containing a single integration site. Systematic mutation of the central att site dinucleotide revealed that cleavage was independent of nucleotide sequence, but rejoining was crucially dependent upon complementarity of the cleavage products. Recombination between att sites containing dinucleotides with antiparallel complementarity led to antiparallel recombination. Integrase-substrate pre-incubation experiments revealed that the enzyme can form an attP-integrase tetramer complex that then captures naked attB DNA, and suggested that two alternative assembly pathways can lead to synaptic complex formation.

[1]  D. Filman,et al.  A structural basis for allosteric control of DNA recombination by λ integrase , 2005, Nature.

[2]  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.

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

[4]  D. Warren,et al.  A chimeric Cre recombinase with regulated directionality , 2008, Proceedings of the National Academy of Sciences of the United States of America.

[5]  T. Kieser.  Practical streptomyces genetics , 2000 .

[6]  W. Ross,et al.  The λ phage att site: functional limits and interaction with Int protein , 1980, Nature.

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

[8]  Eric C. Olivares,et al.  A phage integrase directs efficient site-specific integration in human cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

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

[10]  S. Woo,et al.  Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cDNA , 2005, Proceedings of the National Academy of Sciences of the United States of America.

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

[12]  A. Bradbury,et al.  Using an in vivo phagemid system to identify non‐compatible loxP sequences , 2001, FEBS letters.

[13]  G R Stark,et al.  Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[14]  I. Whang,et al.  Analyses of the first chemical step in Flp site-specific recombination: Synapsis may not be a pre-requisite for strand cleavage. , 1996, Journal of molecular biology.

[15]  Michele P Calos,et al.  Phage integrases: biology and applications. , 2004, Journal of molecular biology.

[16]  Xiaoming Ding,et al.  Highly Efficient In Vitro Site-Specific Recombination System Based on Streptomyces Phage φBT1 Integrase , 2008, Journal of bacteriology.

[17]  Graham F Hatfull,et al.  Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase , 2006, Nature Methods.

[18]  Milind Gupta,et al.  Sequences in attB that affect the ability of ϕC31 integrase to synapse and to activate DNA cleavage , 2007, Nucleic acids research.

[19]  L. Leinwand,et al.  A genetic screen identifies novel non-compatible loxP sites. , 2002, Nucleic acids research.

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

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

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

[23]  N. Grindley,et al.  Catalytic residues of gamma delta resolvase act in cis. , 1995, The EMBO journal.

[24]  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.

[25]  W. Stark,et al.  Sin recombinase from Staphylococcus aureus: synaptic complex architecture and transposon targeting , 2002, Molecular microbiology.

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

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

[28]  J. Hartley,et al.  DNA cloning using in vitro site-specific recombination. , 2000, Genome research.

[29]  D. Gidoni,et al.  FLP-mediated recombination for use in hybrid plant production. , 2000, The Plant journal : for cell and molecular biology.

[30]  N. Craig,et al.  The mechanism of phage λ site-specific recombination: Site-specific breakage of DNA by Int topoisomerase , 1983, Cell.

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

[32]  H. Nash,et al.  Synapsis of attachment sites during lambda integrative recombination involves capture of a naked DNA by a protein-DNA complex , 1988, Cell.

[33]  H. Nash,et al.  Nicking-closing activity associated with bacteriophage lambda int gene product. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

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