Prior to initiating DNA synthesis, Escherichia coli oriC switches from ORC, comprising initiator DnaA bound at three high-affinity sites, to pre-RC, when additional DnaA molecules interact with low-affinity sites. Two types of low-affinity sites exist: R boxes that bind DnaA-ATP and DnaA-ADP with equal affinity, and I-sites with a three- to fourfold preference for DnaA-ATP. To assess the regulatory role of weak DnaA interactions during pre-RC assembly in vivo, we compared the behaviour of plasmid-borne wild-type oriC with mutants having an increased or decreased number of DnaA-ATP discriminatory I-sites. Increasing the number of discriminatory sites by replacing R5M with I2 inactivated extrachromosomal oriC function. Mutants with no discriminatory sites perturbed host growth and rapidly replaced wild-type chromosomal oriC, but normal function returned if one I-site was restored at either the I2, I3 or R5M position. These observations are consistent with assembly of E. coli pre-RC in vivo from mixtures of DnaA-ATP and DnaA-ADP, with I-site interactions coupling pre-RC assembly to DnaA-ATP levels.