DNA-binding parameters of the HU protein of Escherichia coli to cruciform DNA.
We have previously studied the binding characteristics of the HU protein of Escherichia coli to different linear DNAs. In this work, using gel-retardation and footprint analysis, we studied the specific binding of HU protein to a synthetic cruciform DNA. We have quantified our results in order to precisely define the binding and cooperativity constants of HU protein towards cruciform DNA and compare them to those obtained with linear DNA. We used stringent high-salt conditions versus non-stringent low-salt conditions in order to differentiate the non-specific-protein HU-DNA complexes from the specific, high-salt-resistant complexes. We observe that HU-protein dimers bind specifically to the cruciform DNA with a binding constant K = 2.0 x 10(8) M-1 and a value for the cooperativity constant omega = 1 corresponding to a non-cooperative phenomenon. For the first time we observe a footprint pattern of HU protein bound to DNA using the hydroxyl-radical-footprinting technique on HU-protein-cruciform-DNA complexes. The residues protected by HU protein are localized at and near the junction point but interestingly they are mainly present in two of the four oligonucleotides which constitute the cruciform DNA. These two oligonucleotides are unpaired and opposite each other. These results support a model where two HU-protein dimers specifically bind to two equivalent angles present opposite each other in the four-way-junction-DNA structure with almost no dimer-dimer interactions.
Bonnefoy
E
E
Institut de Biologie Physico-chimique, Paris, France.
Takahashi
M
M
Yaniv
J R
JR
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C040330:histone-like protein HU, bacteria; D004247:DNA