Nuclear magnetic resonance comparison of the binding sites of mithramycin and chromomycin on the self-complementary oligonucleotide d(ACCCGGGT)2. Evidence that the saccharide chains have a role in sequence specificity.
A comprehensive two-dimensional 1H nuclear magnetic resonance spectral analysis of the ternary 4:2:1 mithramycin-Mg2+-d(A1C2C3C4G5G6G7T8)2 complex and the ternary 2:1:1 chromomycin-Mg(2+)-d(A21C2C3C4C4G5G6G7T8)2 complex is presented. The self-complementary oligonucleotide is found to bind two dimers of mithramycin in two identical off-center binding sites such that the twofold symmetry of the oligonucleotide is retained. In contrast, the same oligonucleotide binds only one dimer of chromomycin in a single but distinct off-center binding site. Two-dimensional nuclear Overhauser spectroscopy experiments show that the aglycone binding site of the drug dimer in each complex extends over almost four base-pairs and is similar in length to other complexes between chromomycin or mithramycin and oligonucleotides. The data demonstrate that the chromomycin dimer binding site is offset by one base-pair step from the dimer binding site in the mithramycin complex. This preferred binding site prevents two dimers of chromomycin binding to d(ACCCGGGT)2 for steric reasons and lends further support to previous work that showed the 5'-CG base-pair site is less favored by these drugs compared to the 5'GC and 5'-GG,5'-CC sites. Evidence is presented that suggests mithramycin may occupy either of two distinct binding sites on d(ACCCGGGT)2 when the drug concentration is not saturating. The nuclear magnetic resonance data demonstrate that the saccharide chains of this family of drugs do have a role in determining the binding site on nucleotides and as a consequence the CDE trisaccharide chain may alter its conformation to fulfil this role. Titration of mithramycin up to a drug-duplex ratio of 7:1 reveals further association of mithramycin with the complex but no new drug-oligonucleotide nuclear Overhauser enhancement contacts.
Keniry
M A
MA
Research School of Chemistry, Australian National University, Canberra.
Banville
D L
DL
Simmonds
P M
PM
Shafer
R
R
D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002865:Chromomycins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D008926:Plicamycin / Q000378:metabolism*
D002865:Chromomycins; D009838:Oligodeoxyribonucleotides; D008926:Plicamycin