DnaA Protein of Escherichia coli: oligomerization at the E. coli chromosomal origin is required for initiation and involves specific N-terminal amino acids.
Iterated DnaA box sequences within the replication origins of bacteria and prokaryotic plasmids are recognized by the replication initiator, DnaA protein. At the E. coli chromosomal origin, oriC, DnaA is speculated to oligomerize to initiate DNA replication. We developed an assay of oligomer formation at oriC that relies on complementation between two dnaA alleles that are inactive by themselves. One allele is dnaA46; its inactivity at the non-permissive temperature is due to a specific defect in ATP binding. The second allele, T435K, does not support DNA replication because of its inability to bind to DnaA box sequences within oriC. We show that the T435K allele can complement the dnaA46(Ts) allele. The results support a model of oligomer formation in which DnaA box sequences of oriC are bound by DnaA46 to which T435K then binds to form an active complex. Relying on this assay, leucine 5, tryptophan 6 and cysteine 9 in a predicted alpha helix were identified that, when altered, interfere with oligomer formation. Glutamine 8 is additionally needed for oligomer formation on an oriC-containing plasmid, suggesting that the structure of the DnaA-oriC complex at the chromosomal oriC locus is similar but not identical to that assembled on a plasmid. Other evidence suggests that proline 28 of DnaA is involved in the recruitment of DnaB to oriC. These results provide direct evidence that DnaA oligomerization at oriC is required for initiation to occur.
Simmons
Lyle A
LA
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-131, USA.
Felczak
Magdalena
M
Kaguni
Jon M
JM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000483:Alleles; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001704:Biopolymers; D002876:Chromosomes, Bacterial; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D005816:Genetic Complementation Test; D005976:Glutaral / Q000494:pharmacology; D015151:Immunoblotting; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D051738:Origin Recognition Complex; D010957:Plasmids / Q000235:genetics; D021122:Protein Subunits; D018741:Replication Origin; D013696:Temperature; D014764:Viral Proteins
D001426:Bacterial Proteins; D001704:Biopolymers; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D021122:Protein Subunits; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004265:DNA Helicases; D053488:DnaB Helicases; D005976:Glutaral