ACTIVATION OF DISTANT REPLICATION ORIGINS INVIVO BY DNA LOOPING AS REVEALED BY A NOVEL MUTANT FORM OF AN INITIATOR PROTEIN DEFECTIVE IN COOPERATIVITY AT A DISTANCE

We have isolated mutants of the pi-initiator protein of the plasmid R6K that are defective in DNA looping in vitro but retain their normal DNA binding affinity for the primary binding sites (iterons) at the gamma-origin/enhancer. One such looping defective mutant called R6 was determined to be a proline to leucine change at position 46 near the N terminus of the pi-protein. Using a set of genetic assays that discriminate between the activation of the gamma-origin/enhancer from those of the distantly located alpha and beta-origins, we show that the looping defective initiator protein fails to activate the alpha and beta-origins but derepresses initiation from the normally silent gamma-origin in vivo. The results conclusively prove that DNA looping is required to activate distant replication origins located at distances of up to 3 kb from the replication enhancer.