Purification and characterization of HP1 Cox and definition of its role in controlling the direction of site-specific recombination.
The protein that activates site-specific excision of the HP1 genome from the Hemophilus influenzae chromosome, HP1 Cox, was purified. Native Cox consists of four 8.9-kDa protomers. Tetrameric Cox self-associates to octamers; the apparent dissociation constant was 8 microM protomer, suggesting that under reaction conditions Cox is largely tetrameric. Cox binding sites consist of two direct repeats of the consensus motif 5'-GGTMAWWWWA; one Cox tetramer binds to each motif. Cox binding interfered with the interaction of HP1 integrase with one of its binding sites, IBS5. This competition is central to directional control, as shown by studies on mutated sites. Both Cox binding sites were necessary for Cox to fully inhibit integration and activate excision, although Cox continued to affect recombination when the single binding site proximal to IBS5 remained intact. Eliminating the IBS5 site completely prevented integration but greatly enhanced excision. Excisive recombination continued to require Cox even when IBS5 was inactivated. Cox must therefore play a positive role in assembling the nucleoprotein complexes producing excisive recombination, by inducing the formation of a critical conformation in those complexes.
Esposito
D
D
Department of Biochemistry, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205, USA.
Scocca
J J
JJ
D000595:Amino Acid Sequence; D017101:Bacteriophage P1 / Q000235:genetics*; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006193:Haemophilus influenzae / Q000235:genetics* / Q000821:virology; D019426:Integrases / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification*
C070354:Cox protein, Enterobacteria phage P2; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060509:HP1 integrase; D004247:DNA; D019426:Integrases