PURIFICATION AND CHARACTERIZATION OF THE INTEGRASE FROM THE HAEMOPHILUS-INFLUENZAE BACTERIOPHAGE-HP1 - IDENTIFICATION OF A 4-STRANDED INTERMEDIATE AND THE ORDER OF STRAND EXCHANGE

The integrase encoded by the temperate phage HPI promotes the site-spe cific recombination between DNA sites on its genome (the attP site) an d on the genome of the host Haemophilus influenzae (the affB site). Th e protein has been overproduced in Escherichia coli, and purified to a pparent homogeneity. HP1 integrase promotes recombination of supercoil ed attP-containing molecules with linear segments with attB sites. Rea ction was enhanced by spermidine and by the bacterial DNA-bending prot ein integration host factor. The rate of recombination showed complex and related dependence upon the integrase concentration and the concen tration of the supercoiled attP substrate. These relationships probabl y originate from the need to assemble a multi-protein complex on the a ttP DNA. The reaction promoted by HP1 integrase produced a four-strand ed initial reaction product in which one pair of DNA strands had under gone 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 com ponent had the kinetic and structural properties of an intermediate in the recombination reaction. The existence of this intermediate was us ed to determine that the two strand exchanges required for recombinati on of the duplex substrates proceed in a defined order.