Purification and characterization of bacteriophage P22 Xis protein.
The temperate bacteriophages lambda and P22 share similarities in their site-specific recombination reactions. Both require phage-encoded integrase (Int) proteins for integrative recombination and excisionase (Xis) proteins for excision. These proteins bind to core-type, arm-type, and Xis binding sites to facilitate the reaction. lambda and P22 Xis proteins are both small proteins (lambda Xis, 72 amino acids; P22 Xis, 116 amino acids) and have basic isoelectric points (for P22 Xis, 9.42; for lambda Xis, 11.16). However, the P22 Xis and lambda Xis primary sequences lack significant similarity at the amino acid level, and the linear organizations of the P22 phage attachment site DNA-binding sites have differences that could be important in quaternary intasome structure. We purified P22 Xis and studied the protein in vitro by means of electrophoretic mobility shift assays and footprinting, cross-linking, gel filtration stoichiometry, and DNA bending assays. We identified one protected site that is bent approximately 137 degrees when bound by P22 Xis. The protein binds cooperatively and at high protein concentrations protects secondary sites that may be important for function. Finally, we aligned the attP arms containing the major Xis binding sites from bacteriophages lambda, P22, L5, HP1, and P2 and the conjugative transposon Tn916. The similarity in alignments among the sites suggests that Xis-containing bacteriophage arms may form similar structures.
Mattis
Aras N
AN
Department of Pathology, University of California, San Francisco, San Francisco, California, USA.
Gumport
Richard I
RI
Gardner
Jeffrey F
JF
D017100:Bacteriophage P22 / Q000201:enzymology* / Q000235:genetics; D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002850:Chromatography, Gel; D018983:DNA Footprinting; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004279:DNA, Viral; D014764:Viral Proteins; D004254:DNA Nucleotidyltransferases; C063911:excisionase