title: Mutant DnaAs of Escherichia coli that are refractory to negative control.
abstract: 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 substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression 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 replication. 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 initiation in Escherichia coli.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA and Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA.
authors.forename: Sundari
authors.initials: S
authors.lastname: Chodavarapu
authors.forename: Magdalena M
authors.initials: MM
authors.lastname: Felczak
authors.forename: Lyle A
authors.initials: LA
authors.lastname: Simmons
authors.forename: Alec
authors.initials: A
authors.lastname: Murillo
authors.forename: Jon M
authors.initials: JM
authors.lastname: Kaguni
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D018741:Replication Origin
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C083980:dnaN protein, Bacteria; D000251:Adenosine Triphosphatases; C435211:Hda protein, E coli