Mutant DnaAs of Escherichia coli that are refractory to negative control

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine sub-stitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (b clamp), and both DnaAcos and H202Y resist inhib-ition by the Hda-b clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensi-tive or refractory to inhibition by the Hda-b clamp complex in vitro but are responsive to hda expres-sion in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA repli-cation. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initi-ation in Escherichia coli