The integration host factor ofEscherichia colibinds to multiple sites at plasmid R6K γ origin and is essential for replication

Abstract

Examination of the effect of the himA and himD mutants of Escherichia coli on the maintenance of plasmid R6K has revealed that the .gamma. origin-containing replicons cannot be established in any of the mutants deficient in the production of E. coli Integration Host Factor (IHF). Contrary, the R6K derivatives containing other origins of the plasmid (.alpha. and/or .beta.) replicate in a host lacking functional IHF protein. We show that IHF protein binds specifically to a segment of the replication region which is essential for the activity of all three R6K origins. Mapping the IHF binding sequence with neocarzinostatin showed that the protein protects three segments of the origin: two strong binding sites reside within an AT-rich block, while the third, considerably weaker site is separated from the other two by a cluster of the seven 22 bp direct repeats. These seven repeats have been shown previously to bind the R6K-encoded initiator protein .pi.. We also demonstrate that the establishment of .pi.-origin complexes prior to IHF addition prevents the binding of the IHF protein to the .gamma. origin. The binding sequence of IHF and .pi. proteins do not overlap; therefore, we propose that the binding of .pi. protein alters the structure of the DNA and thereby prevents the subsequent binding of IHF protein.