Keith R. Fox

Kinetics of dissociation of nogalamycin from DNA: comparison with other anthracycline antibiotics

Stopped-flow spectrometry and simple mixing techniques have been employed to investigate the detergent-induced dissociation of anthracycline antibiotics from natural and synthetic DNAs. Both daunomycin and nogalamycin dissociate more slowly from poly(dG-dC) than from poly(dA-dT) but the difference is much more marked for nogalamycin. With an equimolar mixture of poly(dG-dC) and poly(dA-dT), or with poly(dA-dC).poly(dG-dT), dissociation of nogalamycin occurs very slowly. In all cases the release of antibiotic from a synthetic polynucleotide is a one-step process following a single exponential. Dissociation of daunomycin, adriamycin and iremycin from calf thymus DNA is a more complex reaction which requires a two-exponential fit, in contrast to earlier reports, but differences between the behaviour of the three antibiotics are minor. Dissociation of nogalamycin from natural DNA requires a three-exponential fit, is in general far slower, and depends upon the base composition, the level of binding and the time allowed for the complex to equilibrate. It is concluded that sequence selectivity is minimal or lacking for daunomycin, whereas nogalamycin binding is sequence dependent and probably involves migration of the antibiotic between DNA binding sites. There is an inverse correlation between dissociation rate constants and antibacterial potency in simple tests.

Evidence of different binding sites for nogalamycin in DNA revealed by association kinetics

The kinetics of association between nogalamycin and DNA have been measured by stopped-flow spectrometry. With a naturally occurring DNA (calf thymus) the reaction profile requires not less than three exponentials for its complete description. By contrast, binding to poly(dA-dT) is fully described by two exponentials which correspond to the two faster components seen with the natural DNA, whereas binding to poly(dG-dC) is a single exponential process whose time constant is about the same as the slowest component measured with calf thymus DNA. In all cases the amplitude of each component in the decay varies considerably with polynucleotide concentration. The results are consistent with a model in which the antibiotic is only able to bind directly to regions of the DNA which are transiently perturbed, probably non-basepaired. As a result, the antibiotic interacts much faster with AT-rich rather than GC-rich DNA sequences, which may provide a basis for its apparent sequence selectivity.

The use of micrococcal nuclease as a probe for drug-binding sites on DNA

The cutting pattern produced by micrococcal nuclease on three DNA fragments has been determined in the absence and presence of various DNA-binding drugs. The enzyme itself cuts almost exclusively at pA and pT bonds, showing a greater activity at (A-T)n than in homopolymeric runs of A and T. Each drug produces distinct changes in the cleavage pattern. The protected regions can not be pinpointed with sufficient precision to assess the exact drug-binding sites on account of the sequence selectivity of the enzyme, although where a direct comparison is possible these include most of those seen as DNAase I footprints. The enzyme is most useful for assessing the selectivity of drugs which bind to AT-rich regions. Several drugs protect the DNA from micrococcal nuclease attack in regions which do not contain their acknowledged best binding sites. It appears that micrococcal nuclease is sensitive to the existence of secondary drug-binding sites which are not evident with other footprinting techniques.

Kinetics of dissociation of quinoxaline antibiotics from DNA

The kinetics of detergent-induced dissociation of triostins A and C and quinomycin C from DNA have been investigated. All three antibiotics dissociate from poly(dA-dT) and poly(dG-dC) in a simple first-order fashion whereas their dissociation from a natural DNA (calf thymus) is complex, requiring three exponential terms for its complete description. This behaviour is attributed to sequence-selectivity on the part of the drugs and seems to represent dissociation from different classes of intercalative binding site. The time constants of dissociation are better resolved for quinomycins than for triostins, consistent with the view that quinomycins are more sequence-specific in their interaction with DNA, but it is not possible to identify any class of binding site with the alternating purine-pyrimidine sequences of the synthetic polydeoxynucleotides. In general, the triostins dissociate an order of magnitude faster than the corresponding quinomycins. This is attributable to a larger entropy of activation, presumably reflecting greater flexibility of the octapeptide ring when the cross-bridge is a disulphide as opposed to the slightly shorter thioacetal found in quinomycins. The longest time constant in the dissociation of each of the four quinoxaline antibiotics from calf thymus DNA correlates well with its antibacterial potency, in agreement with the conclusion that the biological effects result from impairment of the role of DNA as a template for polymerase activity.

Molecular Aspects of the Interaction between Quinoxaline Antibiotics and Nucleic Acids

Quinoxaline antibiotics are of widespread occurrence in nature. They are produced by a variety of species of streptomycetes and are characterised by an octadepsipeptide ring to which are attached two moieties of quinoxaline-2-carboxylic acid, hence their name. They were first isolated in the 1950s (see Katagiri et al., 1975 for a full description) and the best-known member of the group is echinomycin (Corbaz et al., 1957; Keller-Schierlein et al., 1959) whose structure, shown in figure 1, was recently revised.

Equilibrium and kinetic studies on the binding of des-N-tetramethyltriostin A to DNA.

The interaction between TANDEM (a des-methyl analogue of triostin A) and poly(dA-dT) results in extension of the helix by 6.8 A for each ligand molecule bound, exactly as predicted for a bis-intercalation reaction. Cooperativity is evident in Scatchard plots for the interaction at ionic strengths of 0.2 and 1.0, where the binding constant is diminished compared to that which pertains at low salt concentrations. Binding to a natural DNA (calf thymus), already considerably weaker than binding to poly(dA-dT), is also sensitive to increased ionic strength. With a self-complementary octanucleotide d(G-G-T-A-T-A-C-C) the binding curve indicates the presence of a single des-N-tetramethyltriostin A binding site per helical fragment with a non-cooperative association constant about 6 . 10(6) M-1. Detergent-induced dissociation of des-N-tetramethyltriostin A-poly(dA-dT) complexes results in a simple exponential decay at all levels of binding, but the time constant of decay is dependent upon the initial binding ratio. This behavior cannot directly explain the cooperativity of equilibrium binding isotherms but suggests the occurrence of relatively long-lived perturbations of the helical structure by binding of the ligand. [Ala3, Ala7]des-N-tetramethyltriostin A, which has a more flexible octapeptide ring lacking the disulphide cross-bridge, dissociates from poly(dA-dT) much faster than des-N-tetramethyltriostin A. Dissociation of des-N-tetramethyltriostin A from calf thymus DNA is more rapid than dissociation of triostin A or other quinoxaline antibiotics, which may account for its low antimicrobial activity.

Differential inhibition of a restriction enzyme by quinoxaline antibiotics

The inhibition of cleavage by HpaI at two well-defined restriction sites in linearised phi X174-RF DNA by quinoxaline antibiotics has been investigated. Echinomycin, which displays a certain preference for binding to GC basepairs, inhibits cleavage at one site much more than the other, whereas triostin A, which displays less pronounced sequence-selectivity, inhibits both sites about equally. Other congeners inhibit reaction at the two sites with varying effectiveness. The results demonstrate the usefulness of studying inhibition of cleavage at specific sites by restriction enzymes as a means of exploring the specificity of DNA-ligand interactions.