Characterization of the binding sites of two proteins involved in the bacteriophage P2 site-specific recombination system.
Integration of the bacteriophage P2 genome into the Escherichia coli host chromosome occurs by site-specific recombination between the phage attP and E. coli attB sites. The phage-encoded 38-kDa protein, integrase, is known to be necessary for both phage integration as well as excision. In order to begin the molecular characterization of this recombination event, we have cloned the int gene and overproduced and partially purified the Int protein and an N-terminal truncated form of Int. Both the wild-type Int protein and the integration host factor (IHF) of E. coli were required to mediate integrative recombination in vitro between a supercoiled attP plasmid and a linear attB substrate. Footprint experiments revealed one Int-protected region on both of the attP arms, each containing direct repeats of the consensus sequence TGTGGACA. The common core sequences at attP and attB were also protected by Int from nuclease digestion, and these contained a different consensus sequence, AA T/A T/A C/A T/G CCC, arranged as inverted repeats at each core. A single IHF-protected site was located on the P (left) arm, placed between the core- and P arm-binding site for Int. Cooperative binding by Int and IHF to the attP region was demonstrated with band-shift assays and footprinting studies. Our data support the existence of two DNA-binding domains on Int, having unrelated sequence specificities. We propose that P2 Int, IHF, attP, and attB assemble in a higher-order complex, or intasome, prior to site-specific integrative recombination analogous to that formed during lambda integration.
Yu
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Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
Haggård-Ljungquist
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D001426:Bacterial Proteins / Q000378:metabolism; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004279:DNA, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D019426:Integrases; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D011485:Protein Binding; D011995:Recombination, Genetic; D016662:Virus Integration
D001426:Bacterial Proteins; D004279:DNA, Viral; D034982:Integration Host Factors; D004254:DNA Nucleotidyltransferases; D019426:Integrases