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

EMBO J. 2001 Mar 15;20(6):1469-76. doi: 10.1093/emboj/20.6.1469.


The initiator protein DnaA of Escherichia coli binds to a 9mer consensus sequence, the DnaA box (5'-TT(A/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 that binding to ATP-DnaA boxes in the AT-rich region of oriC of E.coli requires 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 the single-stranded region, thereby stabilizing it. This demonstrates an additional binding specificity of DnaA protein to single-stranded ATP-DnaA boxes. Binding affinities, as judged by the DnaA concentrations required for site protection in footprinting, were approximately 1 nM for DnaA box R1, 400 nM for double-stranded ATP-DnaA boxes and 40 nM for single-stranded ATP-DnaA boxes, respectively. We propose that sequential recognition of high- and low-affinity sites, and binding to single-stranded origin DNA may be general properties of initiator proteins in initiation complexes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / metabolism
  • Adenosine Triphosphate / metabolism
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Binding Sites
  • DNA Footprinting
  • DNA Replication*
  • DNA, Bacterial / biosynthesis*
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / metabolism*
  • Escherichia coli / genetics*
  • Models, Genetic
  • Molecular Sequence Data
  • Protein Binding
  • Replication Origin*
  • Surface Plasmon Resonance


  • Bacterial Proteins
  • DNA, Bacterial
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • DnaA protein, Bacteria
  • Adenosine Diphosphate
  • Adenosine Triphosphate