Irving H. Goldberg

Base Specificity in the Interaction of Polynucleotides with Antibiotic Drugs

Echinomycin, daunomycin, ethidium bromide, nogalamycin, chromomycin, mithramycin, and olivomycin inhibit RNA synthesis by RNA polymerase by interacting with the DNA template. Chromomycin and olivomycin form complexes with DNA, preferably in the helical form, but not with RNA. These complexes require guanine in DNA and the addition of a stoichiometric amount of bivalent cation. None of the other antibiotics requires the presence of any single base in the template for its action.

Actinomycin and nucleic acid function.

Publisher Summary The chapter discusses the current understanding of actinomycin action at the molecular level. This discussion is related particularly to a model recently proposed for the reaction of actinomycin with DNA and to the implications of this model for the template function of helical nucleic acids. Actinomycin blocks the expression of genetic potentialities in several ways. The most prominent of these involves suppression of the formation of the primary cellular gene products; the ribonucleic acids (RNAs). The antibiotic also affects gene action, by preventing the transport of gene products, from nucleus to cytoplasm. In some mammalian cells, exposure to actinomycin is associated with the breakdown of previously synthesized RNA and this may constitute a third form of interference, with the gene action, although such depolymerization of RNA may merely reflect the existence of a normal turnover cycle. The actinomycin has also been applied to the analysis of gene action and the role of RNA metabolism in many systems. The chapter also reviews briefly some of these areas. It has not been attempted to include all the relevant contributions, but was tried to cite representative studies that illustrate the nature of actinomycin action, within a particular experimental framework, or exemplify some current applications of the antibiotic to various biological problems and the kind of information it can help to obtain.

Actinomycin D Inhibition of Deoxyribonucleic Acid-Dependent Synthesis of Ribonucleic Acid

Minute amounts of actinomycin D inhibit the synthesis of ribonucleic acid by nuclear extracts of HeLa cells in a ribonucleic acid-synthesizing system that is dependent on deoxyribonucleic acid and requires the presence of all four ribonucleoside triphosphates. The inhibition can be reversed by adding deoxyribonucleic acid to the enzymatic reaction. These findings support the work of others on the mode of action of actinomycin D in vivo.

Neocarzinostatin: Spectral characterization and separation of a non-protein chromophore

Abstract Chromatographically purified neocarzinostatin exhibits absorption, fluorescence, magnetic circular dichroic and circular dichroic spectral characteristics above and below 300 nm atypical for a protein with its reported aminoacid composition, indicating the presence of a non-protein chromophore. The drug complex, stable at acidic pH, can be dissociated by treatment with reducing or denaturing agents at neutral or basic pH. Chromatography of the dissociated complex, or more conveniently, methanol extraction of the lyophilized drug, separates a protein with an amino-acid composition identical to neocarzinostatin and a highly fluorescent chromophore free of amino-acids.

DNA strand scission by the antitumor protein neocarzinostatin.

Abstract The antibiotic protein, neocarzinostatin, induces the scission of DNA strands in vivo and in vitro . HeLa cell DNA prelabelled with [ 14 C] thymidine is cut into large pieces with a peak at 80–90S when cells are incubated with 0.5 to 5.0 μg/ml of highly purified neocarzinostatin. Incubation of the antibiotic (0.5 μg/ml) with [ 3 H] SV40 DNA in the presence of 2-mercaptoethanol results in the conversion of superhelical DNA I to nicked circular duplex DNA II. At high levels of drug, smaller fragments of linear DNA are produced. Strand breaks are detected in both neutral and alkaline sucrose gradients, indicating that drug susceptibility is not due to alkali-labile bonds.

The relationship between DNA strand-scission and DNA synthesis inhibition in HeLa cells treated with neocarzinostatin

Neocarzinostatin inhibits DNA synthesis in HeLa S3 cells and induces the rapid limited breakage of cellular DNA. The fragmentation of cellular DNA appears to precede the inhibition of DNA synthesis. Cells treated with drug at 37 degrees C for 10 min and then washed free of drug show similar levels of inhibition of DNA synthesis or cell growth, or of strand-scission of DNA as when cells were not washed. If cells are preincubated with neocarzinostatin at 0 degrees C before washing, the subsequent incubation of 37 degrees C results in no inhibition of DNA synthesis or cell growth, or cutting of DNA. Isolated nuclei or cell lysates derived from neocarzinostatin-treated HeLa S3 cells are inhibited in DNA synthesis but this can be overcome in cell lysates by adding activated DNA. A cytoplasmic fraction from drug-treated cells can stimulate DNA synthesis by nuclei isolated from untreated cells, whereas nuclei from drug-treated cells are not stimulated by the cytoplasmic fraction from untreated cells. By contrast, neocarzinostatin does not inhibit DNA synthesis when incubated with isolated nuclei, but it can be shown that under these conditions the DNA is already degraded and is not further fragmented by the drug. These data suggest that the drugs ability to induce breakage of cellular DNA in HeLa S3 cells is an essential aspect of its inhibition of DNA replication and may be responsible for the cytotoxic and growth-inhibiting actions of neocarzinostatin.

An effect of pactamycin on the initiation of protein synthesis in reticulocytes

Low levels of pactamycin (about 10−6 M) stop polypeptide synthesis by intact reticulocytes and their lysates after a lag of about two minutes. This effect which is similar to that of poly A, an inhibitor of polypeptide chain initiation, is not additive to that of poly A. Cells which have been pretreated with NaF and must form new polypeptide chains are more sensitive to pactamycin than cells which are able to complete pre-existing chains. High levels of pactamycin (⩾10−5 M) inhibit elongation in addition to initiation. 3H-pactamycin binds to 80S ribosomes and the 40S subunits at 0°. Monosomes produced by NaF treatment of reticulocytes bind 3H-pactamycin very effectively, whereas those produced by RNase treatment of polyribosomes do not.

Single-strand nicking of DNA in vitro by neocarzinostatin and its possible relationship to the mechanism of drug action

Neocarzinostatin, a protein antibiotic with anti-tumor activity was found to place single-strand scissions in DNA in an in vitro reaction. The drugs cutting activity was strongly dependent on the presence of 2-mercaptoethanol or dithiothreitol but some cutting did take place in the absence of reducing agent at very high drug levels and prolonged incubation. The requirement for reducing agents could not be replaced with NAD+, FAD, NADH or H2O2 and the strand-scission reaction was not affected by Mg2+, EDTA or intercalating agents. Similar profiles of heat-inactivation of neocarzinostatin were found whether activity was measured by the scission of DNA strand either in vitro or in HeLa cells treated with the drug. Furthermore, both of these parameters corresponded closely with the ability of the modified drug to inhibit DNA synthesis and growth of HeLa cells. By column isoelectric focusing it was shown that all four activities are associated with the same protein band (pH 3.28). From these data we conclude that the cytotoxic activity of neocarzinostatin and the nicking of DNA strands in vitro appear to reside in the same protein.

Neocarzinostatin chromophore: Presence of a highly strained ether ring and its reaction with mercaptan and sodium borohydride

Spectroscopic evidence suggests the presence of a highly strained ether ring (Fig. 1) (possibly an epoxide) in the C12-subunit of the previously determined partial structure 2a (Fig. 2) of the major neocarzinostatin chromophore (NCS-Chrom A) which completes assignment of all the oxygens in the molecule. The main product from mercaptan treatment suggests opening of the ether ring involving the addition of one molecule of mercaptan as well as reduction of the C12-substructure, whereas a parallel two-step reduction occurs on NaBH4 treatment. Both reactions occur with rearrangement of the C12-substructure and the implication for the mechanism of action of NCS-Chrom A in DNA strand scission activity is discussed. The evidence suggests a downward revision of the molecular formula for NCS-Chrom A as well as minor components B and C by two protons.

Solution structure of a two-base DNA bulge complexed with an enediyne cleaving analog.

Nucleic acid bulges have been implicated in a number of biological processes and are specific cleavage targets for the enediyne antitumor antibiotic neocarzinostatin chromophore in a base-catalyzed, radical-mediated reaction. The solution structure of the complex between an analog of the bulge-specific cleaving species and an oligodeoxynucleotide containing a two-base bulge was elucidated by nuclear magnetic resonance. An unusual binding mode involves major groove recognition by the drug carbohydrate unit and tight fitting of the wedge-shaped drug in the triangular prism pocket formed by the two looped-out bulge bases and the neighboring base pairs. The two drug rings mimic helical DNA bases, complementing the bent DNA structure. The putative abstracting drug radical is 2.2 ± 0.1 angstroms from the pro-S H5′ of the target bulge nucleotide. This structure clarifies the mechanism of bulge recognition and cleavage by a drug and provides insight into the design of bulge-specific nucleic acid binding molecules.