Binding Of ActD Research Articles

Investigating actinomycin D binding to G-quadruplex, i-motif and double-stranded DNA in 27-nt segment of c-MYC gene promoter

c-MYC DNA is an attractive target for drug design, especially for cancer chemotherapy. Around 90% of c-MYC transcription is controlled by NHE III1, whose 27-nt purine-rich strand has the ability to form G-quadruplex structure. In this investigation, interaction of ActD with 27-nt G-rich strand (G/c-MYC) and its equimolar mixture with the complementary sequence, (GC/c-MYC) as well as related C-rich oligonucleotide (C/c-MYC) was evaluated. Molecular dynamic simulations showed that phenoxazine and lactone rings of ActD come close to the outer G-tetrad nucleotides indicating that ActD binds through end-stacking to the quadruplex DNA. RMSD and RMSF revealed that fluctuation of the quadruplex DNA increases upon interaction with the drug. The results of spectrophotometry and spectrofluorometry indicated that ActD most probably binds to the c-MYC quadruplex and duplex DNA via end-stacking and intercalation, respectively and polarity of ActD environment decreases due to the interaction. It was also found that binding of ActD to the GC-rich DNA is stronger than the two other forms of DNA. Circular dichroism results showed that the type of the three forms of DNA structures doesn't change, but their compactness alters due to their interaction with ActD. Finally, it can be concluded that ActD binds differently to double stranded DNA, quadruplex DNA and i-motif.

The structural basis of actinomycin D–binding induces nucleotide flipping out, a sharp bend and a left-handed twist in CGG triplet repeats

The potent anticancer drug actinomycin D (ActD) functions by intercalating into DNA at GpC sites, thereby interrupting essential biological processes including replication and transcription. Certain neurological diseases are correlated with the expansion of (CGG)n trinucleotide sequences, which contain many contiguous GpC sites separated by a single G:G mispair. To characterize the binding of ActD to CGG triplet repeat sequences, the structural basis for the strong binding of ActD to neighbouring GpC sites flanking a G:G mismatch has been determined based on the crystal structure of ActD bound to ATGCGGCAT, which contains a CGG triplet sequence. The binding of ActD molecules to GCGGC causes many unexpected conformational changes including nucleotide flipping out, a sharp bend and a left-handed twist in the DNA helix via a two site-binding model. Heat denaturation, circular dichroism and surface plasmon resonance analyses showed that adjacent GpC sequences flanking a G:G mismatch are preferred ActD-binding sites. In addition, ActD was shown to bind the hairpin conformation of (CGG)16 in a pairwise combination and with greater stability than that of other DNA intercalators. Our results provide evidence of a possible biological consequence of ActD binding to CGG triplet repeat sequences.

Force spectroscopy reveals the DNA structural dynamics that govern the slow binding of Actinomycin D

Actinomycin D (ActD) is a small molecule with strong antibiotic and anticancer activity. However, its biologically relevant DNA-binding mechanism has never been resolved, with some studies suggesting that the primary binding mode is intercalation, and others suggesting that single-stranded DNA binding is most important. To resolve this controversy, we develop a method to quantify ActD’s equilibrium and kinetic DNA-binding properties as a function of stretching force applied to a single DNA molecule. We find that destabilization of double stranded DNA (dsDNA) by force exponentially facilitates the extremely slow ActD-dsDNA on and off rates, with a much stronger effect on association, resulting in overall enhancement of equilibrium ActD binding. While we find the preferred ActD–DNA-binding mode to be to two DNA strands, major duplex deformations appear to be a pre-requisite for ActD binding. These results provide quantitative support for a model in which the biologically active mode of ActD binding is to pre-melted dsDNA, as found in transcription bubbles. DNA in transcriptionally hyperactive cancer cells will therefore likely efficiently and rapidly bind low ActD concentrations (∼10 nM), essentially locking ActD within dsDNA due to its slow dissociation, blocking RNA synthesis and leading to cell death.

Multiple Binding Modes of Actinomycin D Reveal the Basis for its Potent HIV-1 and Cancer Activity

Actinomycin D (ActD) is a well studied anti-cancer agent that is used as a prototype for developing new generations of drugs. However, the biophysical basis of its activity is still unclear. Because ActD is known to intercalate double stranded DNA (dsDNA), it was assumed to block replication by stabilizing dsDNA in front of the replication fork. However, recent studies have shown that ActD binds with even higher affinity to imperfect duplexes and some sequences of single stranded DNA (ssDNA). These features suggest that ActD may alternatively destabilize complementary dsDNA. In this work we use optical tweezers to stretch and relax single dsDNA molecules in the presence of varying ActD concentrations. We observe that ActD binds with highest affinity to two separate DNA strands that are connected by ActD. This binding mode is ∼1000-fold stronger than ActD's intercalation into dsDNA. We are able to characterize at least two classes of ActD-ssDNA binding sites that differ in dissociation times (∼10% of sites with ∼1000 sec off time, and the rest with ∼10 sec off time). The much weaker ActD binding to dsDNA relative to ssDNA leads to duplex destabilization, in contrast to conventional intercalation. At saturation, the ActD-dsDNA complex becomes indistinguishable from the saturated ActD-ssDNA. These results suggest that two separate, anti-parallel DNA strands constitute the highest affinity natural substrate for ActD binding, with Kd ∼10-100 nM and a relatively slow off rate. This finding supports the hypothesis that the primary characteristic of ActD that contributes to its biological activity is its ability to inhibit cellular replication by stabilizing DNA bubbles during RNA transcription, thereby stalling the transcription process.

Novel Molecular Mechanism for Actinomycin D Activity as an Oncogenic Promoter G-Quadruplex Binder

Actinomycin D (ActD) is a natural antibiotic that inhibits the transcription of genes by interacting with a GC-rich duplex, a single-stranded or hairpin form of DNA, and then interfering with the action of RNA polymerase. In this study, we identified a novel molecular mechanism of anticancer activity of ActD as an oncogenic c-Myc promoter G-quadruplex binder. ActD selectively inhibits the elongation of oligonucleotides containing c-Myc promoter G-quadruplex sequence in PCR-stop assays. UV-vis spectroscopic and circular dichroism studies suggest that ActD interacts with c-Myc promoter G-quadruplex via a surface end stacking interaction, inducing a mixed-type conformation of the G-quadruplex. ActD selectively inhibits the cellular growth and synthesis of c-Myc mRNA in Ramos cells having the NHEIII(1) region in the translocated c-Myc gene. In addition, the results of promoter assays using two kinds of NHEIII(1) region mutants and wild-type constructs strongly support the idea that binding of ActD with G-quadruplex formed in the promoter region results in the reporter gene being turned off. Our study reveals a novel mechanism underlying the anticancer activity of ActD, whereby ActD interacts with oncogenic promoter G-quadruplex DNA to repress gene expression.

Interaction of Actinomycin D with Promoter Element of c-met and Its Inhibitory Effect on the Expression of c-Met

c-Met, the tyrosine kinase receptor for hepatocyte growth factor plays a pivotal role in normal cellular signaling and overexpression of c-Met protein is reported in several human cancers. Thus, transcriptional regulation of c-met appears to be an attractive target for chemotherapy. Therefore, we selected a 24mer GC rich sequence (24R) from the c-met promoter located at −142 to −119 from transcription start site and studied its interaction with anticancer drug, Actinomycin D. Spectroscopic analysis demonstrated a strong complexation between ActD and 24RY as shown by: (i) a high binding constant, K of 4–5 × 105 M−1 with ΔΔG of −47 ± 1.5 Kcalmol−1; (ii) marked increase by + 10 °C in melting temperature of 24RY; and (iii) significant changes in circular dichroic spectra of both ActD and 24RY. Molecular modeling revealed the preference of ActD to the Spl binding site, GGCGGG, in 24RY. Expression of the c-Met was checked in HepG2 cells, a human hepatocellular carcinoma cell line by using western blotting and immunocytochemistry. Downregulation of c-Met expression by as much as 50% was observed in the presence of 20ng/ml (IC50) of ActD. Taking into account of the binding studies also, we feel that the down regulation of c-Met perhaps involves binding of ActD to the promoter site of c-met. Therefore, c-met could be a challenging and promising target for therapeutic strategies in combating cancer.

Distinguishing Dual DNA Binding Modes of Actinomycin D using Optical Tweezers

Actinomycin D (ActD), the first antibiotic which exhibited anti-tumor activity, was initially believed to bind double stranded DNA (dsDNA) through intercalation. Later it was shown to bind single stranded DNA (ssDNA) with an order of magnitude higher affinity. ssDNA binding can be extremely important in inhibiting replication of viruses that replicate through ssDNA templates such as HIV. While these two binding modes can be separately quantified by studying binding to specific substrates, it is very difficult to determine the mode of binding to polymeric DNA. DNA stretching studies can precisely quantify intercalation by measuring the increase in DNA length upon intercalation. However, ssDNA binding also increases DNA length. Therefore, we have developed a method that combines the measured increase in DNA length with the overall DNA melting free energy change, allowing us to simultaneously determine ssDNA binding and intercalation as DNA is stretched. Using this method, we were able to distinguish between dual binding modes of ActD. We determined that the ssDNA binding of ActD (Kss ∼ 108 M−1) is much higher than its binding to dsDNA (Kds ∼ 106 M−1) for long polymeric DNA. We also determined the ssDNA and dsDNA binding site size, which are 3 bases and 6 base pairs, respectively.

Solution structure of the ActD-5'-CCGTT3GTGG-3' complex: drug interaction with tandem G.T mismatches and hairpin loop backbone.

Binding of actinomycin D (ActD) to the seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3 GTGG-3' has been studied in solution using high-resolution nuclear magnetic resonance (NMR) techniques. A strong binding constant (8 x 10(6) M(-1)) and high quality NMR spectra have allowed us to determine the initial DNA structure using distance geometry as well as the final ActD-5'-CCGTT3 GTGG-3' complex structure using constrained molecular dynamics calculations. The DNA oligomer 5'-CCGTT3GTGG-3' in the complex forms a hairpin structure with tandem G.T mismatches at the stem region next to a loop of three stacked thymine bases pointing toward the major groove. Bipartite T2O-GH1 and T2O-G2NH2 hydrogen bonds were detected for the G.T mismatches that further stabilize this unusual DNA hairpin. The phenoxazone chromophore of ActD intercalates nicely between the tandem G.T mismatches in essentially one major orientation. Additional hydrophobic interactions between the ActD quinoid amino acid residues with the loop T5-T6-T7 backbone protons were also observed. The hydrophobic G-phenoxazone-G interaction in the ActD-5'-CCGTT3GTGG-3' complex is more robust than that of the classical ActD- 5'-CCGCT3GCGG-3' complex, consistent with the roughly 2-fold stronger binding of ActD to the 5'-CCGTT3GTGG-3' sequence than to its 5'-CCG CT3GCGG-3' counterpart. Stabilization by ActD of a hairpin containing non-canonical stem base pairs further strengthens the notion that ActD or other related compounds may serve as a sequence- specific ssDNA-binding agent that inhibits human immunodeficiency virus (HIV) and other retroviruses replicating through ssDNA intermediates.

Crystal structure of actinomycin D bound to the CTG triplet repeat sequences linked to neurological diseases.

The potent anticancer drug actinomycin D (ActD) acts by binding to DNA GpC sequences, thereby interfering with essential biological processes including replication, transcription and topoisomerase. Certain neurological diseases are correlated with expansion of (CTG)n trinucleotide sequences, which contain many contiguous GpC sites separated by a single base pair. In order to characterize the binding of ActD to CTG triplet repeat sequences, we carried out heat denaturation and CD analyses, which showed that adjacent GpC sequences flanking a T:T mismatch are preferred ActD-binding sites, and that ActD binding results in a conformational transition to A-type structure. The structural basis of the strong binding of ActD to neighboring GpC sites flanking a T:T mismatch was provided by the crystal structure of ActD bound to ATGCTGCAT, which contains a CTG triplet sequence. Binding of two ActD molecules to GCTGC causes a kink in the DNA helix. In addition, using a synthetic self-priming DNA model, 5'-(CAG)4(CTG)(16)-3', we observed that ActD can trap the cruciform or duplexes of (CTG)n and interfere with the expansion process of CTG triplet repeats as shown by gel electrophoretic expansion assay. Our results may provide the possible biological consequence of ActD bound to CTG triplet repeat sequences.

Crystallographic analysis of a novel complex of actinomycin D bound to the DNA decamer CGATCGATCG.

The potent anticancer drug actinomycin D (ActD) acts by binding to DNA, thereby interfering with replication and transcription. ActD inhibits RNA polymerase far more specifically than DNA polymerase. Such discrimination is not easily understood by the conventional DNA binding mode of ActD. We have solved and refined at 1.7 A resolution the crystal structure of ActD complexed to CGATCGATCG, which contains no canonical GpC binding sequence. The crystal data are space group P4(3)2(1)2, a = b = 47.01 A, and c = 160.37 A. The structure was solved by the multiple wavelength anomalous diffraction method using a 5-bromo-U DNA. The asymmetric unit of the unit cell contains two independent dimers of a novel slipped duplex complex consisting of two decamer DNA strands bound with two ActD drug molecules. (The DNA in one dimer is numbered C1 to G10 in one strand and C11 to G20 in the complementary strand and in the second dimer, C101 to G110 and C111 to G120, respectively.) The structure reveals a highly unusual ActD binding mode in which the DNA adopts a slipped duplex with the A3-T4/A13-T14 dinucleotides looped out. ActD intercalates between G2-C11* (C11* being from a symmetry-related molecule) and C5-G20 base pairs. Two such slipped duplex-ActD complexes bound to each other by mutually intercalating their T4/T14 bases into the helix cavities (located between C5-G20 and G6-C19 base pairs) of neighboring complexes, forming a dimer of drug-DNA complexes. The binding site mimics the drug binding at the elongation point during transcription. Modeling studies show that the ActD-DNA complex fits snugly in the active site cavity in RNA polymerase but not in DNA polymerase. This may explain the strong preference of ActD inhibition toward transcription.

Water regulation of actinomycin-D binding to DNA: the interplay among drug affinity, DNA long-range conformation, and hydration.

Actinomycin-D (actD) binds to natural DNA at two different classes of binding sites, weak and strong. The affinity for these sites is highly dependent on DNA sequence and solution conditions, and the interaction appears to be purely entropic driven. Although the entropic character of this reaction has been attributed to the release of water molecules upon drug to DNA complex formation, the mechanism by which hydration regulates actD binding and discrimination between different classes of binding sites on natural DNA is still unknown. In this work, we investigate the role of hydration on this reaction using the osmotic stress method. We show that the decrease of solution water activity, due to the addition of sucrose, glycerol, ethylene glycol, and betaine, favors drug binding to the strong binding sites on DNA by increasing both the apparent binding affinity delta G, and the number of DNA base pairs apparently occupied by the bound drug nbp/actD. These binding parameters vary linearly with the logarithm of the molar fraction of water in solution log(chi w), which indicates the contribution of water binding to the energetic of the reaction. It is demonstrated that the hydration change measured upon binding increases proportionally to the apparent size of the binding site nbp/actD. This indicates that nbp/actD, measured from the Scatchard plot, is a measure of the size of the DNA molecule changing conformation due to ligand binding. We also find that the contribution of DNA deformation, gauged by nbp/actD, to the total free energy of binding delta G, is given by delta G = delta Glocal + nbp/actD x delta GDNA, where delta Glocal = -8020 +/- 51 cal/mol of actD bound and delta GDNA = -24.1 +/- 1.7 cal/mol of base pair at 25 degrees C. We interpret delta Glocal as the energetic contribution due to the direct interactions of actD with the actual tetranucleotide binding site, and nbp/actD x delta GDNA as that due to the change in conformation, induced by binding, of nbp/actD DNA base pairs flanking the local site. This interpretation is supported by the agreement found between the value of delta GDNA and the torsional free energy change measured independently. We conclude suggesting an allosteric model for ligand binding to DNA, such that the increase in binding affinity is achieved by increasing the relaxation of the unfavorable free energy of binding storage at the local site through a larger number of DNA base pairs. The new aspect on this model is that the "size" of the complex is not fixed but determined by solutions conditions, such as water activity, which modulate the energetic barrier to change helix conformation. These results may suggest that long-range allosteric transitions of duplex DNA are involved in the inhibition of RNA synthesis by actD, and more generally, in the regulation of transcription.

Actinomycin D Amplifies Site-Specific DNA Cleavage Induced by Neocarzinostatin

Neocarzinostatin (NCS) is an antineoplastic antibiotic causing DNA cleavage. Actinomycin D (ActD) is an antineoplastic antibiotic, which does not cleave DNA strands. We examined the mechanism of ActD-mediated amplification of NCS-induced DNA cleavage using32P-labeled DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene. NCS plus glutathione caused DNA cleavage at thymine and adenine residues in the absence of ActD. The addition of ActD enhanced the DNA cleavage at the sites at which the lesions on opposite strands are staggered 2 bases in a 3′ direction, particularly at the 5′-TCT-3′·3′-AGA-5′, 5′-TGT-3′·3′-ACA-5′ and 5′-ACT-3′·3′-TGA-5′ sequences, suggesting that ActD amplified double-stranded DNA cleavage. The mechanism of ActD-mediated amplification of NCS-induced DNA cleavage can be explained by assuming that binding of ActD to DNA changes the DNA conformation to allow NCS to bind to DNA at the specific sequences.

Is the strong actinomycin D binding of d(5'CGTCGACG3') the consequence of end-stacking?

It has been reported that ACTD binds strongly and cooperatively to a non-GC containing self-complementary octamer d(CGTCGACG) with a 2:1 drug to duplex ratio (Synder et al., 1989). If one views the classic intercalative preference of ACTD for the 5'GpC3' sequence to be the drug favoring the 3'-side of dG, the possibility exists that the drug molecules may in fact stack on the G.C base pairs at both ends of this oligomeric duplex. To investigate this possibility, d(CGTCGACG) and several related oligomers resulting from replacing the terminal base(s) or appending with dT and/or dA are used in a comparative study employing equilibrium titration, thermal denaturation, kinetic, and various spectral measurements. Absorbance titrations at 20 degrees C confirm the strong and highly cooperative nature of ACTD binding to this octamer. The stoichiometric association constants for the binding of the first and second drugs were found to be 1 x 10(5) and 3.2 x 10(7) M-1, respectively. The base replacements of dG and dC at the respective ends resulted in a much weaker ACTD binding affinity, the loss of binding cooperativity, and much faster association and dissociation kinetics. These are consistent with the inability of the drug to stack on the 3'-side of dG due to base replacements. Appending the end(s) with dA and/or dT resulted in some diminution of binding affinity and cooperativity, appearance of slower association kinetic components, and unusually strong 7-amino-ACTD fluorescence enhancement for oligomers with dA or dT attached to dG at the 3'-terminal. To further support our postulate, studies were also made with d(CGACGTCG), which is related to the parent octamer by inverting the A.T pairs. It was found that, despite the altered internal sequence, this oligomer exhibits cooperative ACTD binding and kinetic characteristics very similar to those of the parent octamer, consistent with its ability to end-stack on the 3'-side of dG.

Formation of multiple complexes between actinomycin D and a DNA hairpin: structural characterization by multinuclear NMR.

The solution conformations of a DNA oligomer and its complexes with the anticancer drug actinomycin D (ActD) were characterized using homo- and heteronuclear NMR techniques. Previous high-resolution NMR investigations of ActD-DNA complexes employed symmetric double-stranded DNA oligomers, yielding two identical symmetry-related complexes. In order to understand the important effects that neighboring base pairs and/or unusual nucleic acid structures may have on ActD binding specificity and orientation, we chose to study the oligonucleotide d(TCGCGTTTTCGCGA), which adopts a hairpin structure in solution. NOE cross-peak intensities were used to generate distance constraints for molecular dynamics simulations and structure determinations of the free oligonucleotide and for both complexes. A total of 86 intermolecular NOEs were identified for each complex, 27 of which involve exchangeable protons. These intermolecular NOEs along with changes in the phosphorus chemical shifts were used to determine the drug binding site on the DNA. As expected, ActD intercalated exclusively at the single d(GC) step in the DNA hairpin. Interestingly, although the two complexes, which differ by the orientation with which the asymmetric drug chromophore intercalates the DNA, were not formed in equal concentrations, their conformations are very similar. The RMS difference of the DNA hairpin in the two complexes is only 1.10 A. The structures of the minor groove binding pentapeptide rings are not affected by any of the changes in the normal double-helical structure imposed by the hairpin loop. The total pairwise RMS difference over all atoms for the four peptides (two per complex) in the calculated structures is 0.72 A. Conversely, the structure of the hairpin loop is not appreciably changed upon binding--the RMS difference between the free DNA loop region and the loop region in the two complexes is 1.68 A and only 0.43 A between the two complexes. Our data also support a possible conformation of the d(T)4 loop that does not possess a thymine-thymine "wobble" base pair.

Site-specific binding constants for actinomycin D on DNA determined from footprinting studies

We report site-specific binding constants for the intercalating anticancer drug actinomycin D (Act-D), binding to a 139-base-pair restriction fragment from pBR 322 DNA. The binding constants are derived from analysis of footprinting experiments, in which the radiolabeled 139-mer is cleaved using DNase I, the cleavage products undergo gel electrophoresis, and, from the gel autoradiogram, spot intensities, proportional to amounts of cleaved fragments, are measured. A bound drug prevents DNase I from cleaving at approximately 7 bonds, leading to decreased amounts of corresponding fragments. With the radiolabel on the 3' end of the noncoding strand (A-label), we measured relative amounts of 54 cleavage products at 25 Act-D concentrations. For cleavage of the 139-mer with the label on the 3' end of the coding strand (G-label), relative amounts of 43 cleavage products at 11 Act-D concentrations were measured. These measurements give information about approximately 120 base pairs of the restriction fragment (approximately 12 turns of the DNA helix); in this region, 14 strong and weak Act-D binding sites were identified. The model used to interpret the footprinting plots is derived in detail. Binding constants for 14 sites on the fragment are obtained simultaneously. It is important to take into account the effect of drug binding at its various sites on the local concentration of probe elsewhere. It is also necessary to include in the model weak as well as strong Act-D sites on the carrier DNA which is present, since the carrier DNA controls the free-drug concentration. As expected, the strongest sites are those with the sequence (all sequences are 5'----3') GC, with TGCT having the highest binding constant, 6.4 x 10(6) M-1. Sites having the sequence GC preceded by G are weak binding sites, having binding constants approximately 1 order of magnitude lower than those of the strong sites. Also, the non-GC-containing sequences CCG and CCC bind Act-D with a binding constant comparable to those of the weak GGC sites. The analysis may reveal drug-induced structural changes on the DNA, which are discussed in terms of the mechanism of Act-D binding.

Binding of actinomycin D to [d(ATCGAT)]2: NMR evidence of multiple complexes

Actinomycin D (actD) binds to the oligonucleotide [d(ATCGAT)]2 with a hypochromatic and red-shifted visible absorbance band compared to free drug and a CD spectrum with double negative bands at 460 and 385 nm. These spectral features are similar to those of the actD-[d(ATGCAT)]2 complex, while actD-[d(AT)5]2 gives spectra similar to those of free drug. Upon dilution or raising the temperature, the spectral characteristics accompanying complex formation disappear in the actD-[(ATCGAT)]2 sample but remain in the actD-[d(ATGCAT)]2 complex under the same experimental conditions. These results suggest that (a) sequence-specific binding of actD occurs with [d(ATCGAT)]2 but not with [d(AT)5]2, (b) the binding is not as strong as with [d(ATGCAT)]2, and (c) actD binds [d(ATCGAT)]2 with the same mechanism as it binds [d(ATGCAT)]2, i.e., by intercalation. From NMR spectra of the actD-[d(ATCGAT)]2 complex, three types of signals can be detected below 20 degrees C, one major and two minor ones. At higher temperatures, exchange between the two minor ones becomes fast enough that only one type of minor signal was seen. Partial resonance assignments were made by using 2D nuclear Overhauser effect (NOE) and 2D homonuclear Hartmann-Hahn (HOHAHA) experiments. Proton chemical shift changes of the major complex are consistent with actD chromophore ring intercalation between hexamer base pairs. Data from NOE-detected dipolar interactions between actD and [d(ATCGAT)]2 protons were interpreted in terms of a major complex with the actD chromophore ring system intercalated at the CG position and minor complexes with the drug intercalated off center at the GA positions.(ABSTRACT TRUNCATED AT 250 WORDS)

An NMR investigation of the binding of the anticancer drug actinomycin D to oligodeoxyribonucleotides with isolated 5'd(GC)3' binding sites.

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with one GC binding site [d(ATATGCATAT) (1), d-(ATACGCGTAT) (2), and d(ATATACGCGTATAT) (3)] and with two GC sites [d(ATGCATGCAT) (4)]. At R = 1 (molar ratio of ActD to oligomer duplex) ActD caused a doubling of the number of imino proton signals at, and adjacent to, the GC binding site of 1. One of the G.C base pair signals shifted upfield while the other shifted downfield. Both of the signals for the A.T base pairs adjacent to the binding site shifted downfield. All imino proton signals of 2 and the longer sequence, 3, shifted upfield on binding of ActD to the GC site, indicating a sequence-dependent change in base stacking on complex formation. For both 1 and 2 addition of ActD resulted in a similar pattern of three downfield 31P NMR signals. The two most downfield signals have chemical shift and temperature dependence which are characteristic of phosphate groups at isolated intercalation sites. At R = 1 the ActD complex with 4 has very complex spectra with both upfield and downfield A.T and G.C imino signals. All these data were consistent with two 1:1 complexes with the unsymmetrical phenoxazone ring adopting both of the two possible orientations.(ABSTRACT TRUNCATED AT 250 WORDS)

1H and 31P NMR investigations of actinomycin D binding selectivity with oligodeoxyribonucleotides containing multiple adjacent d(GC) sites.

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with adjacent 5'-GC-3' sites. ActD showed very high specificity for binding to GC sites regardless of oligomer length and surrounding sequence. For a first class of duplexes with a central GCGC sequence, a mixture of 1:1 complexes was observed due to the two different orientations of the ActD phenoxazone ring system. Analysis of 1H chemical shifts suggested that the favored 1:1 complex had the benzenoid side of the phenoxazone ring over the G base in the central base pair of the GCGC sequence. This is the first case in which an unsymmetrical intercalator has been shown to bind to DNA in both possible orientations. A unique 2:1 complex, with significantly different 1H and 31P chemical shifts relative to those of the 1:1 complexes, was formed with these same oligomers, again with the benzenoid side of the ActD molecule over the G base of the central GC base pair. There is considerable anticooperativity to binding of the second ActD in a GCGC sequence. In titrations of oligomers with the GCGC sequence, only the two 1:1 complexes are found up to ratios of one ActD per oligomer. Increasing the ActD concentration, however, resulted in stoichiometric formation of the unique 2:1 adduct. Spectrophotometric binding studies indicated that the apparent binding equilibrium constant for a GC site adjacent to a bound site is reduced by approximately a factor of 20 relative to the ActD binding constant to an isolated GC site.

Actinomycin D in low concentrations binds uniformly to human chromosomes

The binding of actinomycin D (actD) to fixed human metaphase chromosomes was studied by using autoradiography with [ 3H]actD and indirect immunofluorescence with a specific anti-actD antibody. At concentrations of 0.01 and 0.1 μg/ml there was a uniform distribution of drug along the chromosomes as observed by both methods. This is the first study to date characterizing actD binding at such low concentrations to human chromosomes. Since actD intercalates into the DNA helix with GC specificity, our observations indicate that detectable differences in base composition along the lengths of human chromosomes are minimal.