Footprinting studies of DNA-sequence recognition by nogalamycin.

ABSTRACT

We have studied the DNA sequence binding preference of the antitumour antibiotic nogalamycin by DNase-I footprinting using a variety of DNA fragments. The DNA fragments were obtained by cloning synthetic oligonucleotides into longer DNA fragments and were designed to contain isolated ligand-binding sites surrounded by repetitive sequences such as (A)n.(T)n and (AT)n. Within regions of (A)n.(T)n, clear footprints are observed with low concentrations of nogalamycin (< 5 microM), with apparent binding affinities for tetranucleotide sequences which decrease in the order TGCA > AGCT = ACGT > TCGA. In contrast, within regions of (AT)n, the ligand binds best to AGCT; binding to TCGA and TGCA is no stronger than to alternating AT. Within (ATT)n, the preference is for ACGT > TCGA. Although each of these binding sites contains all four base pairs, there is no apparent consensus sequence, suggesting that the selectivity is affected by local DNA dynamic and structural effects. At higher drug concentrations (> 25 microM), nogalamycin prevents DNAse-I cleavage of (AT)n but shows no interaction with regions of (AC)n.(GT)n. Regions of (A)n.(T)n, which are poorly cut by DNase I, show enhanced rates of cleavage in the presence of low concentrations of nogalamycin, but are protected from cleavage at higher concentrations. We suggest that this arises because drug binding to adjacent regions distorts the DNA to a structure which is more readily cut by the enzyme and which is better able to bind further ligand molecules.