500 MHz 1 H‐NMR study of the interaction of daunomycin with B and Z helices of d(CGm 5 CGCG)

Spectroscopic and fluorometric methods were used to study the binding of the anticancer drug daunomycin to poly[d(G-C)] and poly[d(G-m5C)] under a variety of solution conditions. Under high-salt conditions that favor the left-handed Z conformation, binding isotherms for the interaction of the drug with poly[d(G-C)] are sigmoidal, indicative of a cooperative binding process. Both the onset and extent of the cooperative binding are strongly dependent upon the ionic strength. The binding data may be explained by a model in which the drug preferentially binds to B-form DNA and acts as an allosteric effector on the B to Z equilibrium. At 2.4 M NaCl, binding of as little as one drug molecule per 20 base pairs (bp) results in the conversion of poly[d(G-C)] from the Z form entirely to the B form, as inferred from binding data and demonstrated directly by circular dichroism measurements. Similar results are obtained for poly[d(G-m5C)] in 50 mM NaCl and 1.25 mM MgCl2. Under these solution conditions, it is possible to demonstrate the Z to B structural transition in poly[d(G-m5C)] as a function of bound drug by the additional methods of sedimentation velocity and susceptibility to DNase I digestion. The transmission of allosteric effects over 20 bp is well beyond the range of the drug's binding site of 3 bp. Since daunomycin preferentially binds to alternating purine-pyrimidine sequences, which are the only sequences capable of the B to Z transition, the allosteric effects described here may be of importance toward understanding the mechanism by which the drug inhibits DNA replicative events.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Methodscontrasting

confidence: 95%

Long-range allosteric effects on the B to Z equilibrium by daunomycin

Chaires¹

1985

“…First step is a rapid outside binding, followed by intercalation, and it is then followed by conformational changes of either drug, DNA, or both. In addition, high-resolution nuclear magnetic resonance (NMR) studies have been carried out on the binding of daunomycin to DNA, including poly(dA-dT), d(pTpA)3, and d(CGm5CGCG) (Patel & Canuel, 1978; Phillips & Roberts, 1980;Neumann et al, 1985). Daunomycin has also been shown to be a potent effector of the equilibrium between right-handed B-DNA and left-handed Z-DNA (Chaires, 1985).…”

mentioning

confidence: 99%

Interactions between an anthracycline antibiotic and DNA: molecular structure of daunomycin complexed to d(CpGpTpApCpG) at 1.2-.ANG. resolution

Wang¹,

Ughetto²,

Quigley³

et al. 1987

412

283

The crystal structure of a daunomycin-d(CGTACG) complex has been solved by X-ray diffraction analysis and refined to a final R factor of 0.175 at 1.2-A resolution. The crystals are in a tetragonal crystal system with space group P4(1)2(1)2 and cell dimensions of a = b = 27.86 A and c = 52.72 A. The self-complementary DNA forms a six base pair right-handed double helix with two daunomycin molecules intercalated in the d(CpG) sequences at either end of the helix. Daunomycin in the complex has a conformation different from that of daunomycin alone. The daunomycin aglycon chromophore is oriented at right angles to the long dimension of the DNA base pairs, and the cyclohexene ring A rests in the minor groove of the double helix. Substituents on this ring have hydrogen-bonding interactions to the base pairs above and below the intercalation site. O9 hydroxyl group of the daunomycin forms two hydrogen bonds with N3 and N2 of an adjacent guanine base. Two bridging water molecules between the drug and DNA stabilize the complex in the minor groove. In the major groove, a hydrated sodium ion is coordinated to N7 of the terminal guanine and the O4 and O5 of daunomycin with a distorted octahedral geometry. The amino sugar lies in the minor groove without bonding to the DNA. The DNA double helix is distorted with an asymmetrical rearrangement of the backbone conformation surrounding the intercalator drug. The sugar puckers are C1,C2'-endo, G2,C1'-endo, C11,C1'-endo, and G12,C3'-exo. Only the C1 residue has a normal anti-glycosyl torsion angle (chi = -154 degrees), while the other three residues are all in the high anti range (average chi = -86 degrees). This structure allows us to identify three principal functional components of anthracycline antibiotics: the intercalator (rings B-D), the anchoring functions associated with ring A, and the amino sugar. The structure-function relationships of daunomycin binding to DNA as well as other related anticancer drugs are discussed.

“…Additional detail has come from X-ray diffraction studies of fibers of drug-DNA complexes (Pigram et al, 1972), and more recently, a daunomycin dodecamer crystalline complex has yielded resolution of atomic coordinates to 1.2 A (Wang et al, 1987). In parallel with solid-state studies of drug-DNA complexes, there has been an increasing level of detail emerging from NMR analyses of these interactions (Neumann et al, 1985;Luagaa et al, 1986), and this information has been supplemented further by a comparable increase in the application of computer molecular modeling of these systems (Neidle et al, 1987). While all three of these approaches continue to yield a wealth of detail concerning the stereochemistry and dynamics of drug-DNA interactions, they generally require the use of short purified DNA fragments, the sequence of which must be decided in advance of the tThis work was supported by a grant from the Australian Research Grants Scheme. 0006-2960/89/0428-6259S01.50/0 experiment.…”

mentioning

confidence: 99%

Bidirectional transcription footprinting of DNA binding ligands

White¹,

Phillips²

1989

An in vitro transcription assay has been developed to define the exact location of DNA binding ligands. The method employs two counterdirected Escherichia coli promoters separated by approximately 100 bp. Selective transcription from each promoter yields transcripts up to each ligand site. Nonsaturating levels of ligands result in fractional occupancy of ligand at each site, and hence a range of RNA transcript lengths. The bidirectional promoter system results in a transcription footprint which was derived from transcription from both promoters up to the 5' side of each occupied ligand site and defines the sequence specificity and binding site size of the DNA-bound ligand. The transcriptional footprint is precise to +/- 1 bp from the 5' and 3' ends of each binding site. Multiple ligand sites can be ranked in terms of relative fractional occupancy at each site, and the ranking is comparable from either transcription direction. The method was compared to classical DNase I footprinting with a series of DNA binding drugs [actinomycin D, echinomycin, bis(thiadaunomycin), mithramycin, nogalamycin, and an acridine-tripyrrole]. In all cases, specific binding sites were resolved more clearly by transcription footprinting than by DNase I footprinting. Because of the nature of the transcription assay, all occupied ligand sites were detected by this method, in contrast to DNase I footprinting where many sequences are not probed, and where ligand sites are often not accurately defined.