Replication of plasmid R6K gamma origin in vivo and in vitro: dependence on IHF binding to the ihf1 site

Abstract

The gamma origin of plasmid R6K requires the specific initiator protein pi for initiation of replication. However, increased pi concentrations inhibit replication. The host-encoded integration host factor (IHF) protein permits gamma origin replication at otherwise inhibitory pi levels. IHF is thought to mediate this positive effect by directly binding to the gamma origin. In this study we demonstrate that IHF binding to one IHF site in the gama origin, ihf1, but not to the other side, ihf2, is necessary for the gamma origin to replicate at high pi protein levels. We also show that in vitro replication of the gamma origin plasmid requires IHF binding to the ihf1 site. Finally, we demonstrate both in vivo and in vitro that, when mutant pi proteins (hyperactive) are provided instead of wild-type pi, gamma origin plasmids can replicate in the absence of IHF. This supports a previously proposed hypothesis that the pi mutants can bypass the IHF requirement for gamma origin replication.