Crystal and solution structures of 7-amino-actinomycin D complexes with d(TTAGBrUT), d(TTAGTT) and d(TTTAGTTT)

Abstract

The formation of the complex of 7-amino-actinomycin D with potentially single-stranded DNA has been studied by X-ray crystallography in the solid state, by NMR in solution and by molecular modelling. The crystal structures of the complex with 5 H -TTAG[Br 5 U]T-3 H provide interesting examples of MAD phasing in which the dispersive component of the MAD signal was almost certainly enhanced by radiation damage. The trigonal and orthorhombic crystal modi®cations both contain antibiotic molecules and DNA strands in the form of a 2:4 complex: in the orthorhombic form there is one such complex in the asymmetric unit, while in the trigonal structure there are four. In both structures the phenoxazone ring of the ®rst drug intercalates between a BrU±G (analogous to T±G) wobble pair and a G±T pair where the T is part of a symmetry-related molecule. The chromophore of the second actinomycin intercalates between the BrU±G and G±BrU wobble pairs of the partially paired third and fourth strands. The base stacking also involves (A*T)*T triplets and Watson± Crick A±T pairs and leads to similar complex threedimensional networks in both structures, with looping-out of unpaired bases. Although the available NOE constraints of a solution containing the antibiotic and d(TTTAGTTT) strands in the ratio 1:1 are insuf®cient to determine the structure of the complex from the NMR data alone, they are consistent with the intercalation geometry observed in the crystal structure. Molecular-dynamics (MD) trajectories starting from the 1:2 complexes observed in the crystal showed that although the thymines¯anking the d(AGT) core are rather exible and the G±T pairing is not permanently preserved, both strands remain bound to the actinomycin by strong interactions between it and the guanines between which it is sandwiched. Similar strong binding (hemi-intercalation) of the actinomycin to a single guanine was observed in the MD trajectories of a 1:1 complex. The dominant interaction is between the antibiotic and guanine, but the complexes are stabilized further by promiscuous base-pairing.

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

“…It is a neutral molecule that contains a planar tricyclic phenoxazone ring that intercalates dsDNA and two cyclic pentapeptide side chains (Figure 1a). ActD can intercalate between double stranded DNA (dsDNA) base pairs (4–8), bind to single-stranded DNA (ssDNA) (9–12) and can even ‘hemi-intercalate’ between the bases of a single DNA strand (13,14). Early studies found that once bound ActD dissociates slowly from dsDNA (4), with a component of its dissociation occurring on a time scale of ∼1000 s. These studies attributed ActD’s anticancer activity to this slow kinetics, and found it to be due to the slow fitting of its two highly stressed cyclic penta-peptide side chains into the DNA minor groove below and above the intercalated phenoxazone ring (4,15) (Figure 1b).…”

“…The fitting into the groove is stabilized by hydrogen bonding of the ActD side chains to guanine bases (5–7), and associated with major DNA duplex deformations, such as strong bending (6,8), unwinding (6,16) and even base flipping (16,17). Duplex deformations are also driven by optimization of the tricyclic phenoxazone ring stacking with the 3′ faces of guanine (or adenine) residues in the opposite DNA strands (8,14,16). Competing models for the anticancer activity of ActD depend on the favored binding mode; Intercalation may inhibit replication by stabilizing dsDNA in front of the replication fork (8), while binding to destabilized duplexes such as transcription bubbles may inhibit DNA transcription (18–20), and ssDNA binding may directly stall the DNA polymerase (12).…”

Structure of Rhodamine 6G−DNA Complexes from Molecular Dynamics Simulations

“…Structures of chromophore−DNA complexes have been intensively studied in recent years, − because such assemblies may be useful for constructing novel nanoelectronic devices …”

Nucleotide carriers for anti-tumour actinomycin antibiotics

“…This is why the antibiotic increases the DNA melting temperature (19). It is worth noting that, at very high concentrations, the antibiotic intercalates into double helix (24). However, in this case, it acts as a mutagen, similar to ethidium bromide, which unwinds the DNA double helix.…”