A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns.
A biotinylated nucleotide analog containing a disulfide bond in the 12-atom linker joining biotin to the C-5 of the pyrimidine ring has been synthesized. This analog, Bio-SS-dUTP, is an efficient substrate for Escherichia coli DNA polymerase I. Bio-SS-dUTP supported DNA synthesis in a standard nick-translation reaction at 35%-40% the rate of an equal concentration of the normal nucleotide, TTP. DNA containing this analog was bound to an avidin-agarose affinity column and subsequently eluted after reduction of the disulfide bond by dithiothreitol. The ability to recover biotinylated DNA from an avidin affinity column under nondenaturing conditions should prove useful in the isolation of specific protein-DNA complexes. As a demonstration of this approach, Bio-SS-DNA was reconstituted with histones to form 11S monomer nucleosomes. Bio-SS-nucleosomes were shown to selectively bind to avidin-agarose. Ninety percent of the bound Bio-SS-nucleosomes were recovered from the affinity column by elution with buffer containing 50-500 mM dithiothreitol. The recovered nucleosomes were shown to be intact 11S particles as judged by velocity sedimentation in a sucrose gradient. This approach may prove to be generally useful in the isolation of protein-DNA complexes in a form suitable for further analysis of their native unperturbed structure.
D000345:Affinity Labels; D001360:Avidin; D001710:Biotin / Q000031:analogs & derivatives*; D002846:Chromatography, Affinity; D004247:DNA / Q000302:isolation & purification*; D004256:DNA Polymerase I / Q000378:metabolism; D004220:Disulfides; D004926:Escherichia coli / Q000378:metabolism; D009698:Nucleoproteins / Q000302:isolation & purification*; D009707:Nucleosomes / Q000032:analysis; D013379:Substrate Specificity; D014500:Uracil Nucleotides; D014544:Uridine Triphosphate / Q000031:analogs & derivatives*
D000345:Affinity Labels; D004220:Disulfides; D009698:Nucleoproteins; D009707:Nucleosomes; D014500:Uracil Nucleotides; D001360:Avidin; D001710:Biotin; D004247:DNA; D004256:DNA Polymerase I; D014544:Uridine Triphosphate