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 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.
Speck
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Max-Planck-Institut für molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany.
Messer
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011485:Protein Binding; D018741:Replication Origin; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate