DnaA Protein ofEscherichia coli: oligomerization at theE. colichromosomal origin is required for initiation and involves specific N-terminal amino acids

Summary

Iterated DnaA box sequences within the replication origins of bacteria and prokaryotic plasmids are recognized by the replication initiator, DnaA protein. At theE. colichromosomal origin,oriC, DnaA is speculated to oligomerize to initiate DNA replication. We developed an assay of oligomer formation atoriCthat relies on complementation between twodnaAalleles that are inactive by themselves. One allele isdnaA46; 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 withinoriC. We show that theT435Kallele can complement thednaA46(Ts) allele. The results support a model of oligomer formation in which DnaA box sequences oforiCare 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 anoriC-containing plasmid, suggesting that the structure of the DnaA-oriCcomplex at the chromosomaloriClocus 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 tooriC. These results provide direct evidence that DnaA oligomerization atoriCis required for initiation to occur.