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Mechanism of origin unwinding: sequential binding of DnaA to double‐ and single‐stranded DNA
Author(s) -
Speck Christian,
Messer Walter
Publication year - 2001
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/20.6.1469
Subject(s) - dnaa , biology , footprinting , binding site , dna binding protein , dna , biophysics , biochemistry , dna replication , origin of replication , gene , base sequence , transcription factor
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 ∼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.

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