Determination of microscopic binding constants at individual DNA base sequences for the minor groove binders Hoechst 33258, DAPI and pentamidine.
Footprinting titration experiments have been used to estimate microscopic binding constants (KA) for interactions of the ligands Hoechst 33258, DAPI and pentamidine with the 167-mer of the EcoRl-Rsal restriction fragment of the plasmid pBR322. The symmetric base sequence AATTAA yielded the lowest KA values while binding sites which contain an AAA-segment displayed higher binding constants. The accommodation of an adjacent G-C base pair to the ATTT-sequence does not significantly interfere with the stability of the DNA-drug complex. The footprint pattern of the compound Hoechst 33258 shows a peculiarity. Complex formation at low drug concentrations (up to 0.5 mu M) is accompanied by distinct protection sites in the DNA fragment against DNAasel digestion, while at higher drug concentration (up to 5.0 mu M) at the same sites the cleavage activity of the enzyme is enhanced. This is discussed in terms of a conformational change of DNA induced by two concentration-dependent binding modes (AT-specific minor groove binding at low drug concentration and GC-specific interaction at a higher one).
Matesoi
D
D
Institut fur Molekulare Biotechnologie e.V., Jena, Germany.
Kittler
L
L
Bell
A
A
Unger
E
E
Lober
G
G
D001483:Base Sequence; D001665:Binding Sites; D006690:Bisbenzimidazole / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018983:DNA Footprinting; D004262:DNA Restriction Enzymes / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D005456:Fluorescent Dyes / Q000378:metabolism; D007211:Indoles / Q000378:metabolism*; D007364:Intercalating Agents / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D010419:Pentamidine / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism
D005456:Fluorescent Dyes; D007211:Indoles; D007364:Intercalating Agents; C007293:DAPI; D010419:Pentamidine; D004247:DNA; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I; D006690:Bisbenzimidazole