Activation of distant replication origins in vivo by DNA looping as revealed by a novel mutant form of an initiator protein defective in cooperativity at a distance

Abstract

We have isolated mutants of the π 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 γ 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 π protein. Using a set of genetic assays that discriminate between the activation of the γ origin/enhancer from those of the distantly located α and β origins, we show that the looping defective initiator protein fails to activate the α and β origins but derepresses initiation from the normally silent γ 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.