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J. Biol. Chem., Vol. 278, Issue 2, 1005-1011, January 10, 2003
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From the Departments of 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 ~10° opening of the modified base pair toward the
major groove. In contrast, thioguanine significantly altered base pair
dynamics, causing an ~80-fold decrease in the base pair lifetime with
cytosine compared with normal guanine. This perturbation was consistent
with the ~6 °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.
Structure and Dynamics of Thioguanine-modified Duplex
DNA*,
§,§,§,
,§, and§§¶¶
Pharmaceutical Sciences and
Structural Biology, St. Jude Children's Research Hospital,
Memphis, Tennessee 38105, the Departments of
** Pharmaceutical Sciences, § Clinical Pharmacy,
and §§ Molecular Sciences, Health Sciences
Center, University of Tennessee, Memphis, Tennessee 38163, and the
¶ Division of Chemistry, National Center for Toxicological
Research, 3900 NCTR Road, Jefferson, Arkansas 72079
*
This research was supported by American Lebanese Syrian
Associated Charities (ALSAC), a Cancer Center (CORE) Support Grant (CA
21765), The American Cancer Society, and the NCI, National Institutes
of Health Grant R37 CA36401.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Figures
1-3 titled Intrastrand and sequential connectivities between H-1' and
H-6/H-8 protons, Comparison of experimental and back-calculated NMR
spectra, and Comparison of one-dimensional 31P spectra, respectively.
The atomic coordinates and structure factors (1N14 and 1N17 for the G-C and thioG-C DNA duplexes, respectively) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence may be addressed: Dept. of
Pharmaceutical Sciences, St. Jude Children's Research Hospital,
332 N. Lauderdale St., Memphis, TN 38105. Tel.: 901-495-3663;
Fax: 901-495-6869; E-mail: William.Evans@stjude.org.
¶¶
To whom correspondence may be addressed: Dept. of
Structural Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105. Tel.: 901-495-3290; Fax:
901-495-3032; E-mail: Richard.Kriwacki@stjude.org.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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