Solution Structure of a DNA Dodecamer Containing the Anti-Neoplastic Agent Arabinosylcytosine: Combined Use of NMR, Restrained Molecular Dynamics, and Full Relaxation Matrix Refinement

Abstract

The effect of araC incorporation into the dodecamer duplex [d(CGCGAATT) (araC)d(GCG)]2 was examined by comparing its nuclear magnetic resonance (NMR)-determined solution structure with that of the control duplex d[(CGCGAATTCGCG)]2. 1H and 31P resonances in both duplexes were assigned using a combination of 2-D 1H NMR and a 3-D 31P-1H heteroTOCSY-NOESY experiment. Proton-proton distances (determined from NOESY data) and sugar dihedral angles (from NOESY and COSY data) were used in restrained molecular dynamics simulations starting from canonical A- or B-form DNA models. Both the control and araC sets of simulations converged to B-type structures. These structures were subjected to full relaxation matrix refinement to produce final structures which were in excellent agreement (R1/6 < 0.05) with the observed NOE intensities. A detailed comparison of the final control and araC structures revealed a global similarity (overall RMSD approximately 1.3 A), with significant differences localized at the araC site and neighboring bases. These included changes in sugar pucker, backbone torsion angles, base stacking, and other helical parameters. These findings are in general agreement with the previously published X-ray structure of a decamer duplex containing araC. One intriguing feature of the NMR solution structure not found in the crystal structure is the presence of an intramolecular hydrogen bond between the 2' hydroxyl on the araC sugar and the 3' phosphate group.

Mechanisms of apoptosis induction by nucleoside analogs

“…Although the exact nature of the damage caused by nucleoside analogs in stalling replication forks is poorly understood, it follows that cellular responses to these agents are likely to overlap those elicited by drugs such as hydroxyurea (HU) that also cause replication forks to stall by limiting dNTP production. Also, crystallographic studies have demonstrated that incorporation of ara-C into DNA causes localized alterations in the DNA duplex (Schweitzer et al, 1994) and stabilizes covalent topoisomerase I-DNA complexes, thus converting the enzyme into a cellular poison (Pourquier et al, 2000;Chrencik et al, 2003). This suggests that cellular responses to nucleoside analogs are also likely to overlap those elicited by the camptothecin family of topoisomerase I active agents.…”

Structural Impact of the Leukemia Drug 1-ॆ-d-Arabinofuranosylcytosine (Ara-C) on the Covalent Human Topoisomerase I-DNA Complex

“…Two structures of DNA duplexes containing an Ara-C lesion have been reported, one solution structure by nuclear magnetic resonance spectroscopy (NMR) and one crystal structure (17,18). While the atomic coordinates of the crystal structure are not available, we compared the DNA duplex from our Ara-C complex to the 12-base pair DNA duplex containing Ara-C determined by NMR (Fig.…”

“…Even at low concentrations, however, the drug becomes incorporated into DNA and disrupts DNA metabolism (12,17,41). Pourquier et al (12) have shown that the presence of an Ara-C base at the ϩ1 position of the intact strand (opposite the site of single-strand cleavage) slows the rate of DNA strand religation by human topoisomerase I 2-3-fold (12).…”

“…1). Ara-C is thought to inhibit DNA polymerases central to replication and repair processes, and thus to slow the growth of malignant cells (16,17). The detailed impact of Ara-C on human cells, however, is poorly understood.…”

“…The detailed impact of Ara-C on human cells, however, is poorly understood. Incorporation of Ara-C into DNA causes localized alterations in the DNA duplex, including changes in sugar pucker, base stacking, and backbone torsion angles (17,18). Biochemical studies using human topoisomerase I have revealed that Ara-C incorporation at the ϩ1 position of the intact (non-scissile) strand adjacent to the site of singlestrand DNA cleavage induces a 4 -6-fold increase in covalent topoisomerase I-DNA complexes caused by a 2-3-fold decrease in the rate of religation by the enzyme (12).…”