Mechanism of origin unwinding: sequential binding of DnaA to double- and single-stranded DNA

The initiator protein DnaA of Escherichia coli binds to a 9mer consensus se quence, the DnaA box (5'-TTA/(T)TNCACA). If complexed with ATP it adopts a new binding specificity for a 6mer consensus sequence, the ATP-DnaA box (5' -AGatct). Using DNase footprinting and surface plasmon resonance we show th at binding to ATP-DnaA boxes in the AT-rich region of oriC of E. coli requi res binding to the 9mer DnaA box R1, Cooperative binding of ATP-DnaA to the AT-rich region results in its unwinding. ATP-DnaA subsequently binds to th e single-stranded region, thereby stabilizing if. This demonstrates an addi tional binding specificity of DnaA protein to single-stranded ATP-DnaA boxe s. Binding affinities, as judged by the DnaA concentrations required for si te protection in footprinting, were similar to1 nM for DnaA box R1, 400 nM for double-stranded ATP-DnaA boxes and 40 nM for single-stranded ATP-DnaA b oxes, respectively. We propose that sequential recognition of high- and low -affinity sites, and binding to single-stranded origin DNA may be general p roperties of initiator proteins in initiation complexes.