List of journals scanned

Abstract

Bacteriophage λ DNA integrates into the chromosome ofEscherichia coliby first forming an intasome at the phage attachment site on the phage DNA with the integrase Int and integration host factor. This intasome searches the host chromosome for the bacterial attachment site (attB) and then orchestrates two sequential strand exchange reactions to achieve integration. This study characterizes the weak interaction of the intasome andattB. The hypothesis that all of the proteins necessary for integration are brought to the reaction site by the intasome is given additional support by showing that the concentration of phage attachment site and notattB determines the optimal concentration of proteins for integration. The value of the dissocation constant of the complex formed between the intasome andattB is determined in two different ways. First, the rate of the integration reaction is measured as a function of theattB DNA concentration. The saturation constant reflects the dissociation constant of the complex. Second, a recomb ination reaction is inhibited by the inroduction of varying amounts of a secondattB with a sequence change that blocks recombination with this site. The inhibition constant reflects the dissociation constant of the intasome and alteredattB in this experiment. The two methods agree and give a dissociation constant of approximately 300 nM.attB contains two core binding sites for the intasome; it is shown that both are necessary for efficient capture. The value of the dissocation constants are considerably lower when a mutant integrase, IntE174K, is used. This increased affinity for core sites can explain how IntE174K can function in the absence of integration host factor. The inhibition constants also show dependence on the exact sequence of the inhibitingattB. Possible implications of this dependence are discussed.