Characterization of the interaction between the lambda intasome and attB.
Bacteriophage lambda DNA integrates into the chromosome of Escherichia coli by 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 and attB. 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 not attB determines the optimal concentration of proteins for integration. The value of the dissociation constant of the complex formed between the intasome and attB is determined in two different ways. First, the rate of the integration reaction is measured as a function of the attB DNA concentration. The saturation constant reflects the dissociation constant of the complex. Second, a recombination reaction is inhibited by the introduction of varying amounts of a second attB with a sequence change that blocks recombination with this site. The inhibition constant reflects the dissociation constant of the intasome and altered attB 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 dissociation 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 inhibiting attB. Possible implications of this dependence are discussed.
Patsey
R L
RL
Chemistry Department, Vassar College, Poughkeepsie, NY 12601, USA.
Bruist
M F
MF
D001426:Bacterial Proteins / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D019426:Integrases; D034982:Integration Host Factors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011485:Protein Binding; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D004279:DNA, Viral; D034982:Integration Host Factors; D046911:Macromolecular Substances; D004254:DNA Nucleotidyltransferases; D019426:Integrases