Purification and characterization of the integrase from the Haemophilus influenzae bacteriophage HP1; identification of a four-stranded intermediate and the order of strand exchange.
The integrase encoded by the temperate phage HP1 promotes the site-specific recombination between DNA sites on its genome (the attP site) and on the genome of the host Haemophilus influenzae (the attB site). The protein has been overproduced in Escherichia coli, and purified to apparent homogeneity. HP1 integrase promotes recombination of supercoiled attP-containing molecules with linear segments with attB sites. Reaction was enhanced by spermidine and by the bacterial DNA-bending protein integration host factor. The rate of recombination showed complex and related dependence upon the integrase concentration and the concentration of the supercoiled attP substrate. These relationships probably originate from the need to assemble a multi-protein complex on the attP DNA. The reaction promoted by HP1 integrase produced a four-stranded initial reaction product in which one pair of DNA strands had undergone transfer while the other pair remained intact. This four-stranded component was produced more rapidly than any product, and its steady-state level was proportional to the overall rate of reaction. This component had the kinetic and structural properties of an intermediate in the recombination reaction. The existence of this intermediate was used to determine that the two strand exchanges required for recombination of the duplex substrates proceed in a defined order.
Hakimi
J M
JM
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205, USA.
Scocca
J J
JJ
D001287:Attachment Sites, Microbiological / Q000235:genetics; D001426:Bacterial Proteins / Q000494:pharmacology; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006193:Haemophilus influenzae / Q000235:genetics / Q000821:virology*; D019426:Integrases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D034982:Integration Host Factors; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D013379:Substrate Specificity
D001426:Bacterial Proteins; D034982:Integration Host Factors; D011994:Recombinant Proteins; D019426:Integrases