Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells.
OBJECTIVE
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.


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.


CONCLUSIONS
The study implies the possibility of using multiple prokaryotic Int proteins with different specific integration sites in human cells for future gene therapy programmes.
Frumerie
C
C
Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden.
Sylwan
L
L
Helleday
T
T
Yu
A
A
Haggård-Ljungquist
E
E
D000818:Animals; D001287:Attachment Sites, Microbiological; D017901:Bacteriophage P2 / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D002467:Cell Nucleus / Q000821:virology; D004279:DNA, Viral / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000821:virology; D005057:Eukaryotic Cells / Q000378:metabolism / Q000821:virology*; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D019426:Integrases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D011995:Recombination, Genetic; D014162:Transfection / Q000379:methods; D016662:Virus Integration
D004279:DNA, Viral; D019426:Integrases