Correlation of DNA sequence specificity of anthramycin and tomaymycin with reaction kinetics and bending of DNA

Abstract

Anthramycin and tomaymycin are potent antitumor antibiotics belonging to the pyrrolo[1,4]-benzodiazepine [P[1,4]B] group. Their potent biological effects are thought to be due to their ability to react with DNA within the minor groove, forming covalent adducts through the N2 of guanine with the drug molecules overlapping with a 3-4 bp region. In spite of their small molecular weights, the P[1,4]B's show a surprising degree of sequence selectivity, with 5'-PuGPu sequences being the most reactive and 5'-PyGPy sequences being the least reactive [Hertzberg, R. P., Hecht, S. M., Reynolds, V. L., Molineux, I. J., & Hurley, L. H. (1986) Biochemistry 25, 1249-1258]. It has been proposed that inherent DNA flexibility may be one important component of the sequence recognition process for P[1,4]B bonding to DNA, and in this regard, molecular modeling studies are reflective of the experimentally determined hierarchy of bonding sequences [Zakrzewska, K., & Pullman, B. (1986) Biomol. Struct. Dyn. 4, 127-136]. In this study, we have used chemical and enzymatic probes (hydroxyl radical, DNase I) to evaluate drug- and sequence-dependent changes in DNA-adduct conformation, gel electrophoresis to measure drug-induced bending in DNA, and HPLC to measure the reaction kinetics of anthramycin bonding to different sequences. The results show that tomaymycin bonding to DNA induces greater conformational changes in the DNA (i.e., bending and associated narrowing of the minor groove) than anthramycin. In addition, we find that within each drug species (i.e., tomaymycin or anthramycin), sequence specificity correlates with the degree of bending and reaction kinetics such that those sequences with the highest sequence selectivity produce more bending of DNA and react faster with DNA and vice versa. On the basis of these results, we propose that sequence-dependent conformational flexibility may be an important factor in determining the hierarchy of bonding sequences for the P[1,4]B's.