Abstract
Mercaptopurine and thioguanine, two of the most widely used antileukemic agents, exert their cytotoxic, therapeutic effects by being incorporated into DNA as deoxy-6-thioguanosine. However, the molecular mechanism(s) by which incorporation of these thiopurines into DNA translates into cytotoxicity is unknown. The solution structure of thioguanine-modified duplex DNA presented here shows that the effects of the modification on DNA structure were subtle and localized to the modified base pair. Specifically, thioguanine existed in the keto form, formed weakened Watson-Crick hydrogen bonds with cytosine and caused a modest approximately 10 degrees opening of the modified base pair toward the major groove. In contrast, thioguanine significantly altered base pair dynamics, causing an approximately 80-fold decrease in the base pair lifetime with cytosine compared with normal guanine. This perturbation was consistent with the approximately 6 degrees C decrease in DNA melting temperature of the modified oligonucleotide, the 1.13 ppm upfield shift of the thioguanine imino proton resonance, and the large increase in the exchange rate of the thioguanine imino proton with water. Our studies provide new mechanistic insight into the effects of thioguanine incorporation into DNA at the level of DNA structure and dynamics, provide explanations for the effects of thioguanine incorporation on the activity of DNA-processing enzymes, and provide a molecular basis for the specific recognition of thioguanine-substituted sites by proteins. These combined effects likely cooperate to produce the cellular responses that underlie the therapeutic effects of thiopurines.
Highlights
□S The on-line version of this article contains Supplemental Figures 1–3 titled Intrastrand and sequential connectivities between H-1Ј and H-6/H-8 protons, Comparison of experimental and back-calculated NMR spectra, and Comparison of onedimensional 31P spectra, respectively
Previous studies have shown that the cytotoxicity observed in response to thiopurines is a delayed effect associated with inhibition of cell cycle progression through the S and G2 phases, subsequent to the first cell division in which dthioGTP is incorporated into DNA [5, 6]. These findings indicate that thioguanine-containing DNA acts as a poor template for subsequent rounds of DNA replication and that thioG in the template strand is required to elicit the cytotoxic effects of thiopurines
The DNA mismatch repair (MMR) system was initially thought to play a dominant role in mediating the delayed cytotoxic response following thiopurine treatment [9], a recent report indicates that other factors are involved [7]
Summary
The activities of RNase H [5, 12], DNA ligase [8], and topoisomerase II (Topo II) [13], key enzymes involved in DNA repair and replication, have been shown to be significantly altered in the presence of nucleic acid substrates containing single thioG modifications opposite cytosine. These findings show that thioG in the context of a thioG-C base pair modifies the functions of specific DNA-processing and recognition proteins. The goal of the present work was to examine the effects of a single thioG modification opposite cytosine on the structure and dynamics of duplex DNA in solution and to understand the mechanism(s) by which this therapeutically relevant moiety elicits its biological activities
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