Osmium tetroxide probing of local DNA structure in linear and supercoiled plasmids containing curvature-inducing sequences.
Recombinant plasmids pK1A108, pK3A108, pK4A108 and pK5/6T217 containing 80 +/- 1 base pair inserts with different curvature-inducing sequences were studied using the DNA structure probe osmium tetroxide in the presence of pyridine (Os, py). The insertion sequences of the plasmids pK1A108, pK3A108, and pK4A108 are strongly related while the degree of curvature increases from pK1A108 (no curvature) less than pK3A108 less than pK4A108 less than pK5/6T217. The Os, py probe reacts selectively with single-stranded and distorted double-stranded regions in the DNA double helix. Nuclease S1 was used to recognize and cleave regions made permanently single-stranded due to osmium recognize and cleave regions made permanently single-stranded due to osmium modification. In linearized plasmids treatment with Os, py produced no S1-detectable site-specific modification. This result is in agreement with models suggested for DNA curvature; in general, continuous base pairing and base stacking is considered through different sequence blocks as well as through structural junctions. Os, py-probing of the plasmids in the supercoiled state also resulted in no S1-detectable site-specific modification within the inserts of pK1A108, pK3A108, and pK4A108 plasmids (while the regions containing inverted repeat nucleotide sequences in these plasmids were site-specifically modified). In contrast, supercoiled pK5/6T217 DNA was site-specifically modified within the curvature-inducing insert sequence. The nucleotide sequence of the insert of this plasmid strongly differs from the insertion sequences of the other three plasmids; it is extremely AT-rich and contains regularly arranged dAGAGA and dATATA sequences. The structural distortion observed in supercoiled pK5/6T217 is most probably due to the presence of these sequences in a particular arrangement in the insertion sequence.
Palecek
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Institute of Biophysics, Czechoslovak Academy of Sciences, Brno.
Makaturová-Rasovská
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Diekmann
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S
D001483:Base Sequence; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009992:Osmium; D009993:Osmium Tetroxide; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D009992:Osmium; D009993:Osmium Tetroxide