PMID:11250912
| Citation |
Speck, C and Messer, W (2001) Mechanism of origin unwinding: sequential binding of DnaA to double- and single-stranded DNA. EMBO J. 20:1469-76 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC145534 Online version:10.1093/emboj/20.6.1469 |
| Keywords |
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 |
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