Association of Actinomycin D and Deoxyribodinucleotides as a Model for Binding of the Drug to DNA

Abstract

The association of actinomycin D and eleven different deoxyribodinucleotides were studied as model complexes for the interaction of actinomycin D and DNA. All of the deoxyribodinucleotides containing guanine will complex with actinomycin D, and the guanine base has a preferred orientation with respect to the chromophore of actinomycin D (8). The association of actinomycin D and DNA may be monitored by observation of changes in the visible spectrum of actinomycin D when a complex is formed (9). These spectral changes may also be observed by the formation of complexes of actinomycin D and a wide variety of nucleosides and nucleotides (10). Actinomycin D showed a preference for guanosine or guanosine-5'-monophosphate, but the equilibrium constant is 1000 times less than for the interaction of actinomycin D and DNA (10' against 106).Hamilton, Fuller, and Reich (11) proposed that actinomycin D interacts with DNA primarily through hydrogen bond formation, with the chromophore located on the outside of the helix. Muller and Crothers (12) later proposed that the actinomycin D chromophore intercalates into the DNA helix. Sobell et al. (13) have solved the crystal structure of a complex of deoxyguanosine and actinomycin D, and have proposed a model (14, 15) for the actinomycin D-DNA complex that includes both intercalation and hydrogen bond formation. The guanine 2-amino group forms a strong hydrogen bond with the carbonyl oxygen of the L-threonine residue and a weaker hydrogen bond connects the guanine 1911 N(3) ring nitrogen with the N-H group on the iLthreonine residue in this model. The stoichiometry of the complex is one actinomycin D and two deoxyguanosines. In the crystalline complex, the nucleotides and the cyclic pentapeptides are related by an approximate 2-fold symmetry. The importance and possible implications of this 2-fold symmetry have been discussed in detail (5).I have investigated actinomycin D-DNA interaction by studying the association of actinomycin D and various deoxydinucleotides. As will be shown below, the deoxydinucleotides exhibit a great deal of specificity in their interaction with actinomycin D. These experiments support the model proposed by Sobell and Jain (14). MATERIALS AND METHODSActinomycin D was a gift of Merck, Sharpe and Dohme. The deoxydinucleotides were purchased from Collaborative Research, Inc., and, except for pdG-dG, were used without additional purification. The pdG-dG was dissolved in 5 mM phosphate buffer (pH 7.4) and passed through a Millipore filter. Guanosine-5'-monophosphate was purchased from 0 of C CH3

Actinomycin D Binding to Single-stranded DNA: Sequence Specificity and Hemi-intercalation Model from Fluorescence and1H NMR Spectroscopy

“…Both R5 (Figure 2A) and R7 ssDNAs induced significant fluorescent enhancements, as observed previously with D5 and PL7 (Wadkins & Jovin, 1991). Particularly striking was the magnitude of the effect, greater than any reported previously for dsDNA (Gill et al, 1975;Krugh, 1972;Wadkins & Jovin, 1991). The fluorescence spectra of the complexes of R5 and R7 with 7AAMD were very similar to those of D5 (Wadkins & Jovin, 1991), with a shift of the emission maximum from 665 nm (free drug) to 630 nm for the fully bound species.…”

“…The DNAs containing guanine induced a much more pronounced hypochromicity (Figure 1A, B, D), as well as bathochromic shifts, driving the absorption maximum from 505 nm for the free drug to 510 to 540 nm for the complex. This spectral shift is characteristic of stacking interactions between the chromophore of the drug and the bases of DNA, and has been previously noted in the strong binding of 7AAMD to dsDNA (Gill et al, 1975;Graves & Wadkins, 1989;Krugh, 1972).…”

Sequence specificity of mutagen-nucleic acid complexes in solution: Intercalation and mutagen-base pair overlap geometries for proflavine binding to dC-dC-dG-dG and dG-dG-dC-dC self-complementary duplexes

“…5A). This establishes a sequence specificity for complex formation of actinomycin D at purine-(3'-S')-pyrimidine sites at the stable duplex level, in agreement with earlier conclusions at the dinucleoside monophosphate level (20)(21)(22)(23) and tetranucleotide (20,27) and hexanucleotide duplex levels (29).…”

“…There has been considerable interest in the specificity of drug binding to nucleic acid duplexes at the dinucleoside monophosphate (20)(21)(22)(23)(24)(25)(26), self-complementary tetranucleotide (20,27,28), and hexanucleotide (29) duplex level in solution. At the dinucleoside phosphate level, the drug acts as a template on which the nucleic acid forms a miniature double helix (20)(21)(22)(23)(24)(25)(26). Since G+C sequences containing tetranucleotide (mM concentrations) form stable duplexes at low temperature in the absence of drugs, they serve as excellent models for the investigation of drug binding to stable nucleic acid duplexes (20,27,28).…”

The effect of an hypoxic cell sensitizer on tumour growth delay and cell survival. Implications for cell survival in situ and in vitro

“…The antibiotic 1973; Tominaga, Taguchi and Shiba, actinomycin D (ACT), which can bind to 1973). Stewart and Magee (1973), how-DNA and inhibit the DNA dependent ever, have shown that in protein depleted RNA synthesis (Kirk, 1960; Kersten, rats ACT did not modify the incidence of Kersten and Rauen, 1960;Reich et al, dimethylnitrosamine (DMN) 1968 ;Hennings et al, 1968;Threlfall and As a continuation of this experiment, Taylor, 1969;Krugh, 1972), inhibits the the effect of a single treatment with DMN induction of skin tumours in mice by and ACT in different combinations was 7,1 2-dimethylbenz(a)anthracene (DMBA) studied in rats with regard to renal (Gelboin, Klein and Bates, 1965;Hennings carcinogenesis. Additionally, either a and Boutwell, 1967;Bates et al, 1968) normal or a protein deficient diet was fed and also reduces the incidence of mam-since protein depletion is known to in-* Presente(d in part at the Xlth International Cancer Congress, Florence, Italy, October, 1974. fluence the toxicity and the carcinogenic chi-square test, and for the incidence of effect of DMN by inhibition of the drug kidney tumours per animal as well as the metabolizing enzymes in the liver average survival times in the various groups, (McLean and Verschuuren, 1969; McLean the U-test after Mann and Whitney (1947) and .…”