Propagation of pSC101 plasmids defective in binding of integration host factor

Abstract

Integration host factor (IHF), a multifunctional protein of E. coli, normally is required for the replication of plasmid pSC101. T. T. Stenzel, P. Patel, and D. Bastia (Cell 49:709-717, 1987) have reported that IHF binds to a DNA locus near the pSC101 replication origin and enhances a static bend present in this region; mutation of the IHF binding site affects the plasmid's ability to replicate. We report here studies indicating that the requirement for IHF binding near the pSC101 replication origin is circumvented partially or completely by (i) mutation of the plasmid-encoded repA (replicase) gene or the chromosomally encoded topA gene, (ii) the presence on the plasmid of the pSC101 partition (par) locus, or (iii) replacement of the par locus by a strong transcriptional promoter. With the exception of the repA mutation, the factors that substitute for a functional origin region IHF binding site are known to alter plasmid topology by increasing negative DNA supercoiling, as does IHF itself. These results are consistent with the proposal that IHF binding near the pSC101 replication origin promotes plasmid replication by inducing a conformational change leading to formation of a repA-dependent DNA-protein complex. A variety of IHF-independent mechanisms can facilitate formation of the putative replication-initiation complex.