Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins

The gamma origin binding sites of the replication initiator pi protein, composed of seven 22-base-pair (bp) direct repeats and previously shown to be essential for replication of plasmid R6K, can also act as an inhibitor of R6K replication in Escherichia coli cells if provided in trans. Inhibition is dependent upon the ability of these repeats to bind the R6K-encoded pi protein but is not overcome by increasing the intracellular pi level. The insertion of a second repeat cluster in close proximity to the gamma origin also can markedly inhibit replication. The severity of this effect is dependent upon the position, orientation, and number of repeats present in the extra cluster. As few as six extra repeats can result in a completely nonfunctional gamma origin. However, this inactive gamma origin plasmid containing the six extra repeats is functional when placed in a strain that underproduces the wild-type pi protein or when placed in the presence of any of several copy-up mutant pi proteins. On the basis of these observations, we propose that the nucleoprotein structures formed by the binding of pi protein to the seven 22-bp direct repeats at the gamma origin are capable of coupling with each other in vivo and that replication initiation is prevented at such coupled origins. In support of this model of replication control, we demonstrate by electron microscopy analysis that the pi protein has the ability to associate two DNA molecules containing gamma origin sequences and also show that pi enhances the DNA ligase-catalyzed multimerization of a DNA fragment containing the gamma origin.