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DnaA protein of Escherichia coliacts in initiation of chromosomal DNA replication by binding specific sequences, termed DnaA boxes in the chromosomal origin, oriC. On binding, it induces a localized unwinding to create a structure recognized by other replication proteins that act subsequently in the initiation process. In this report, we examined the binding of DnaA protein to each of the DnaA boxes in oriC.By gel mobility shift assays, DnaA protein formed at least six discrete complexes. ATP or ADP included in the reaction mixture prior to electrophoresis was required. Chemical cleavage of isolated complexes with 1,10-phenanthroline-copper revealed that DnaA protein binds in an ordered manner to the DnaA boxes in oriC. Preferential binding to one DnaA box (R4) was confirmed by demonstration that a DNA fragment containing it was bound with greater affinity than another DnaA box sequence (R1). In vitroreplication activity correlated with a complex formed at a ratio of 30 DnaA monomers/oriCin which all DnaA boxes are occupied. The last site bound is DnaA box R3. This event may be critical in promoting initiation from oriCas it correlates with in vivoobservations that binding of DnaA protein to box R3 occurs at the time of initiation of chromosomal replication, whereas other DnaA boxes are bound by DnaA protein throughout the cell cycle (Cassler, M. R., Grimwade, J. E., and Leonard, A. C. (1995) EMBO J.14, 5833-5841).

DnaA protein of Escherichia coliis a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriCplasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA protein-binding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.