STRUCTURAL AND FUNCTIONAL-ANALYSIS OF A REPLICATION ENHANCER - SEPARATION OF THE ENHANCER ACTIVITY FROM ORIGIN FUNCTION BY MUTATIONAL DISSECTION OF THE REPLICATION ORIGIN-GAMMA OF PLASMID R6K

The plasmid R6K possesses three distinct origins of replication: alpha, beta, and gamma. The replication origin-gamma of plasmid R6K performs a dual function: (i) as an origin itself and (ii) as an enhancer element required in cis for the activation at a distance of the other two replication origins-alpha and beta. We have dissected the gamma-origin/enhancer by site-directed mutagenesis and have reached the following conclusions. The origin function can be specifically inactivated without impairing the enhancer function by insertion and/or deletion mutations near the opposite ends of the origin-gamma sequence. One such mutation deleted sequences that included the left DnaA site I. The second mutation involved insertion of linker sequences that resulted in a spatial alteration between the right DnaA site II and the VIIth pi-binding iteron (tandemly repeated binding sites). Other mutations that either partly or completely deleted the A + T-rich sequence adjacent to, but not including, the pi-binding iterons also abrogated enhancer and origin function and suggested that pi-binding sites were necessary but not sufficient for enhancer activity. Finally, the functional analysis of a set of mutants of the gamma-origin/enhancer suggested that a continuous stretch of 300 base pairs is necessary for origin-gamma function and that the sequences that included the binding sites for pi, DnaA, and integration host factor proteins are required in the correct stereochemical alignment to impart origin activity.