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

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.

Co-operative binding of the globular domain of histone H5 to DNA.

The globular domain of histone H5 (GH5) was prepared by trypsin digestion of H5 that was extracted from chicken erythrocyte nuclei with NaCl. Electron microscopy, sucrose gradient centrifugation, native agarose gel electrophoresis and equilibrium density gradient ultracentrifugation show that GH5 binds co-operatively to double-stranded DNA. The electron microscopic images suggest that the GH5-DNA complexes are very similar in structure to co-operative complexes of intact histone H1 (or its variants) with double-stranded DNA, studied previously, which have been proposed to consist of two parallel DNA double helices sandwiching a polymer of the protein. For complexes with GH5 or with intact H1, naked DNA co-sediments with the protein-DNA complexes through sucrose gradients, and DNA also appears to protrude from the ends and sides of the complexes; measurements of the protein-DNA stoichiometry in fractionated samples may not reflect the stoichiometry in the complexes. An estimate of the stoichiometry obtained from the buoyant density of fixed GH5-DNA complexes in CsCl suggests that sufficient GH5 is present in the complexes for the GH5s to be in direct contact, as required by a simple molecular mechanism for the co-operative binding. Chemical crosslinking demonstrates that GH5s are in close proximity in the complexes. In the absence of DNA, GH5-GH5 interactions are weak or non-existent.