Escherichia coli single-stranded DNA binding protein is mobile on DNA: 1H NMR study of its interaction with oligo- and polynucleotides.
The interaction of the Escherichia coli single-stranded DNA binding protein (SSB) with oligo- and poly-nucleotides has been studied by 270-MHz 1H NMR spectroscopy and fast kinetic techniques. d(pT)8 and poly(dT) were used to study noncooperative and cooperative binding, respectively. The H6, H1', and CH3 resonances of d(pT)8 are high-field shifted by less than 0.05 ppm, and H8 and H2 of poly(dA) are low-field shifted upon complexation. The protein resonances remain virtually unshifted. The small shifts upon complexation provide no evidence for extensive stacking interactions between the nucleotide bases and aromatic amino acid side chains of SSB. The d(pT)8 and poly(dA) signals are broadened to about 30 Hz whereas the resonances of poly(dT) are broadened beyond detection upon stoichiometric complexation. Continuous broadening of all poly(dT) signals even at a 10-fold excess of poly(dT) indicates fast exchange of SSB between different binding sites. Dissociation and reassociation rates determined from stopped-flow experiments are too slow by at least 2 orders of magnitude to account for the experimental line widths. Therefore, we conclude that SSB translocates without dissociation from the DNA template. A model for the translocation is outlined. It is based on partial dissociation of octamer sections of poly(dT) from the complex with a rate constant as previously published for the dissociation of d(pT)8 from SSB.
D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000032:analysis*; D009682:Magnetic Resonance Spectroscopy; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D011071:Poly T / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D011089:Polydeoxyribonucleotides; C027903:oligo (dT); D011061:Poly A; D011071:Poly T; C015465:poly(dA)