From the Departments of 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
Received for publication, May 1, 2002, and in revised form, October 17, 2002
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ABSTRACT |
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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.
Mercaptopurine (MP)1 has
been one of the frontline chemotherapeutic agents used to treat
childhood acute lymphoblast leukemia (ALL) since the early 1950s (1).
Presently, both mercaptopurine and thioguanine are among the most
widely used antileukemic agents and are important components of
essentially all modern ALL treatment regimens (2). These thiopurines
are metabolized to deoxy-6-thioguanosine 5'-triphosphate (dthioGTP) via
the purine salvage pathway initiated by hypoxanthine/guanine
phosphoribosyltransferase and subsequently incorporated into DNA (3).
Although incorporation of thiopurines into DNA is required for these
agents to elicit their cytotoxic effects (4, 5) the mechanism(s) by
which DNA incorporation translates into cytotoxicity remains unknown.
Elucidation of these mechanisms will provide new insights into cellular
response to thiopurines and may illuminate novel strategies for
circumventing resistance or enhancing efficacy.
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 (thioG)-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. While incorporation of dthioGTP into DNA leads
to the formation of both thioG-T and thioG-C base pairs (7-9),
in vitro studies have shown that dCTP is incorporated
300-fold more efficiently than deoxythymidine 5'-triphosphate
across a thioG-containing template (7). Therefore, thioG-C base pairs
are likely the most biologically relevant with respect to the
therapeutic effects of thiopurines. Although 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).
Interestingly, several DNA-protein interactions and enzyme activities
involved in DNA replication and repair as well as apoptosis have been
shown to be altered in the presence of thioG-C base pairs. For example,
a protein complex distinct from the MMR complex has been shown to bind
not only thioG opposite T, as does the MMR system (9, 10), but also
thioG opposite C, unlike the MMR system (7). Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), recently implicated as playing roles in DNA
repair as well as apoptosis (11), has been identified as one of the
components of this new protein complex (7). In addition, 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. Importantly, these proteins are components of cellular
response pathways involved in the therapeutic effects of thiopurines.
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.
Sample Preparation--
Single-stranded
oligodeoxyribonucleotides, d(5'-GCTAAGGAAAGCC-3') and the
complementary strand d(5'-GGCTTTCCTTAGC-3'), were synthesized using
standard phosphoramidite chemistry. The thioG-modified oligodeoxyribonucleotide, d(5'-GCTAAGthioGAAAGCC-3'), was
commercially synthesized (Trilink Biotechnologies) using
6-thio-deoyxguanosine-cyanoethyl phosphoramidite (Glen Research).
Single-stranded DNA molecules were purified using anion exchange
chromatography (MonoQ HR 5/5, Amersham Biosciences), as
described by Xu et al. (14). Purity was confirmed by UV
spectroscopy, analytical anion exchange chromatography, and mass
spectrometry. The G-C and thioG-C DNA duplexes (Fig. 1A) were each prepared by
annealing either equimolar amounts of complimentary strands or a 1.2 molar excess of the unmodified strand to the modified strand in
Buffer A (10 mM sodium phosphate, 50 mM NaCl,
pH 7.0) at 70 °C for 5 min, followed by slow cooling over 12 h
to room temperature. DNA duplexes were purified using gel filtration
chromatography (Superdex Peptide HR 10/30, Amersham Biosciences) in
Buffer A.
Thermal Denaturation Studies--
The absorbance of each duplex
DNA (2.3 × 10 NMR Spectroscopy--
NMR spectra were acquired using
either Varian INOVA 600 or 800 MHz spectrometers (Varian NMR Systems)
equipped with 5-mm triple resonance probes with x-,
y-, and z-axis-pulsed magnetic field gradients.
Determination of Imino Exchange Rates--
Imino proton exchange
rates were determined as described by Dhavan et al. (15)
using NMR spectroscopy. Briefly, the method involved selective
inversion of the H2O resonance followed by a variable delay
period, followed finally by detection of imino resonances using
WATERGATE. Longitudinal relaxation times of water were determined
separately using an inversion-recovery pulse sequence (15). Values for
the efficiency of water inversion were determined separately as well.
Data were collected for 1.4 mM G-C and 1.2 mM
thioG-C samples in Buffer A (95% H2O/5% D2O)
at 20 °C at 600 MHz. Base pair lifetimes were determined by
performing the imino proton exchange experiments on 0.9 mM
DNA samples using four different catalyst concentrations at pH 8.1: 10 mM Tris, 45 mM NaCl; 40 mM Tris, 30 mM NaCl; 70 mM Tris, 15 mM NaCl;
100 mM Tris. The imino proton exchange data for G7 in 10 mM Tris could not be interpreted, and therefore was not
included in the determination of the base pair lifetime of G7-C20.
Imino proton exchange rates were obtained by fitting imino resonance
intensities as a function of delay time to standard equations (15)
using the program Kaleidagraph (Adelbeck Software).
NMR Data Processing and Analysis--
NMR experiments were
performed at 20 °C using 1.4 mM G-C and 1.2 mM thioG-C duplex DNA samples in Buffer A. Two- and
three-dimensional NMR spectra were processed using Felix 2000 software
(Molecular Simulations). In general, time domain data was apodized
using sine-bell or shifted sine-bell functions, followed by
zero-filling, and Fourier transformation. 10 mM
sodium 3-trimethylsilyl-proprionate-2,2,3,3,d4 in Buffer
A was used as an external standard. Proton chemical shifts (excluding
H-5', H-5'', and guanine and adenine amino protons) were
assigned using homonuclear two-dimensional NOESY (95%
H2O/5% D2O) and two-dimensional DQF-COSY
(100% D2O) spectra as well as homonuclear
three-dimensional NOESY-TOCSY (100% D2O) spectra (16). Nuclear Overhauser effect (NOE) resonance volumes were extracted from
800 MHz 200-ms and 400-ms two-dimensional NOESY (100% D2O) spectra. A series (100, 200, 300, and 400 ms) of 600 MHz
WATERGATE-NOESY (95% H2O/5% D2O) spectra were
also recorded for qualitative analysis. Upon qualitative assessment of
the G-C and thioG-C 800 MHz DQF-COSY spectra (100% D2O),
H-1'-C-1'-C-2'-H-2' (160 ° ± 30 °) and H-1'-C-1'-C-2'-H-2'' (30 ° ± 30 °) dihedral angle constraints defining a
broad range of pseudorotation angles for the sugar ring were applied to
all residues during the structure calculations. 600-MHz two-dimensional BASHD-TOCSY experiments (17) (100% D2O) were performed to
measure H-3'-31P coupling constants in the absence of
interference from homonuclear proton couplings to H-3'. For those
residues (G-C duplex residues: 1-5, 8-11, 15-24; thioG-C duplex
residues: 2, 3, 7, 9, 11, 15-25) for which a coupling constant could
be determined, the Development of ThioG Force Field
Parameters--
Force field parameters for the nonstandard thioG
nucleoside were derived so as to be consistent with the Cheatham
et al. (19) force field. First, the partial charges for the
HF/6-31G* geometry optimized thioG nucleoside were calculated as
described (20). Bond lengths and angles for the thiocarbonyl of thioG
were obtained from the crystal structures of 6-thiopurine riboside
(21). Force constants were obtained from Miyamoto and Kollman (22).
The Cheatham et al. (19), force field does not include Van
der Waals (VDW) parameters for an sp2 sulfur
found in thiocarbonyl-containing molecules. Therefore, VDW parameters
for sulfur in a thiocarbonyl group were derived. These parameters were
empirically adjusted to best reproduce interaction geometries and
energies between a water molecule and the thioG base as calculated by
high level ab initio calculations (23). The VDW parameters
used in the present work better reproduce the ab initio
calculated geometry of the thioG-C base pair than VDW parameters
previously used to describe thiocarbonyl-containing molecules (22, 24,
25).
Structure Calculations and Analysis--
Restrained molecular
dynamics (rMD) structure refinement of B-DNA starting structures was
performed using an XPLOR 3.1 (26) simulated annealing protocol
employing the Cheatham et al. (19) force field. This force
field was modified to include parameterization of thioG as described
above. Starting structures were energy-minimized by 160 steps of
Powell's conjugate gradient minimization followed by rMD while heating
to 600 K at 50 K sec
A total of 479 and 466 NOE volume constraints were used during the G-C
and thioG-C structure calculations, respectively. These volumes were
interpreted using the full relaxation matrix approach during rMD (28).
Dihedral angle constraints applied were as follows: 26 H-1'-C-1'-C-2'-H-2' and 26 H-1'-C-1'-C-2'-H-2'' for both
structures; 19 C-4'-C-3'-O-P for G-C structures and 16 C-4'-C-3'-O-P for thioG-C structures. Standard purine ring planarity
constraints were applied to both structures. Based on imino proton
assignments and the observation of imino cross-peak resonances
characteristic of standard B-DNA Watson-Crick (W-C) base pairs,
standard B-DNA distance constraints between heavy atoms involved in
hydrogen bonds in W-C base pairs (G-C: O-6-N-4 = 2.91 ± 0.10, N-1-N-3 = 2.95 ± 0.10, N-2-0-2 = 2.86 ± 0.10; A-T: N-1-N-3 = 2.82 ± 0.10, N-6-O-4 = 2.95 ± 0.10) (29) were applied to all base pairs in the G-C and
thioG-C structure calculations except the four terminal base pairs of
each structure and the thioG7-C20 base pair. These constraints were
loosened for the four terminal base pairs (29). The distance
constraints applied to the thioG7-C20 base pair in the first refinement
stage consisted of the standard G-C base pair constraints widened by
0.9 Å. The second stage of refinement was performed using the
following distance constraints for the thioG7-C20 base pair:
S-6-N4 = 3.37 ± 0.10, N-1-N-3 = 3.18 ± 0.10, N-2-O-2 = 2.79 ± 0.10. These distance constraints are a
function of the force field parameters of thioG and were derived from
the AMBER energy-minimized geometry of a thioG-C base pair. Additional standard B-DNA distance constraints between exchangeable protons in W-C
base pairs (30) were applied to both structure calculations except the
four terminal base pairs and the thioG7-C20 base pair, in which cases
these constraints were omitted.
ThioG Decreases the Thermal Stability of Duplex DNA--
The two
molecules studied were both nonself-complementary 13-mer DNA duplexes
(Fig. 1A). The thioG-C
duplex contained a single, centrally positioned thioG modification
(thioG7) opposite C. The unmodified duplex, G-C, contained G at the
central position (G7) along strand 1, opposite C, and was used to
distinguish sequence-specific effects from thioG-induced effects.
Although both duplexes exhibited cooperative thermal denaturation
profiles typical of duplex DNA, the melting temperature of the thioG-C
duplex (Tm = 39.4 °C) was ~6 °C lower
than that of the G-C duplex (Tm = 45.0 °C)
(Fig. 1B). These data suggested that the substitution of S
for O at position 6 in guanine compromised base pair interactions in
the thioG-C duplex relative to those in the G-C duplex (31).
ThioG Has a Local Effect on the Chemical Environment of Duplex
DNA--
Sequential connectivities in two-dimensional NOESY spectra,
including those between anomeric and aromatic protons, were observed for all interior residues of both the G-C and thioG-C duplexes (see
Supplementary Material, Fig. 1 at http://www.jbc.org).
Differences in chemical shifts of each proton of the thioG-C duplex
relative to the corresponding proton of the G-C duplex were recorded.
These data show that thioG had a local effect on the chemical
environment that extended one to two base pairs beyond the thioG
modification (Fig. 1C). However, the most significant change
in the chemical environment occurred at the base pair containing thioG,
thioG7-C20. These data linked the thermal instability of the thioG-C
duplex with localized changes in chemical properties of the thioG7-C20 base pair. The largest difference in proton chemical shifts observed between the two duplexes was that of the imino proton of thioG7 relative to the imino proton of G7 (Fig. 1D). ThioG7 H-1
resonated at 11.38 ppm, 1.13 ppm upfield of the G7 imino proton.
ThioG Exists in the Keto Form in Duplex DNA--
Because bases in
nucleic acids can exist in two tautomeric forms (31), it was possible
that the upfield resonance originally assigned as the thioG7 imino was,
alternatively, the sulfhydryl proton of thioG in the enol tautomeric
form (Fig. 2A). The enol tautomer of thioG is structurally similar to thiophenol. The sulfhydryl proton of thiophenol has been reported to resonate at ~3.5 ppm (www.aist.go.jp/RIODB/SDBS/menu-e.html). This resonance position is
quite different from that of the peak of interest, which resonated at
11.38 ppm. Fig. 2B schematically illustrates protons in
close proximity to the G7 imino proton that should give rise to
cross-peaks in two-dimensional NOESY spectra (32). These cross-peaks
were observed for the G7 imino proton (Fig. 2, C and
E) as well as the proton that resonated at 11.38 ppm in the
thioG-C duplex (Fig. 2, D and F). Together, these
data show that the proton that resonated at 11.38 ppm in the thioG-C
duplex was, in fact, the imino proton of thioG7; therefore, thioG
exists primarily in the keto form within the context of duplex DNA
(Fig. 2A). The large upfield shift of the thioG7 imino
proton resonance relative to the G7 imino proton suggested that the
hydrogen bond formed by thioG7 H-1 and C20 N-3 was significantly
weakened (15, 33) and/or that there may be significant differences in
dynamics between the two DNA duplexes (34). There may also be a
contribution from a through-bond electronic effect due to differences
in the electronegativity and polarizability of S versus O
atoms.
ThioG Modestly Perturbs Duplex DNA Structure--
Since chemical
shift changes are indicative of structural changes, structures of the
G-C and thioG-C DNA duplexes were calculated and compared. As shown in
Table I, the quality of the
resulting ensembles of G-C and thioG-C structures was similar.
Structural analysis showed that both ensembles fell within the B-DNA
family classification and that there were no global differences between the two ensembles of structures (Fig.
3A) as expected.
The effects of thioG on duplex DNA structure that were observed were
subtle and localized to the base pair containing thioG. As depicted in
Fig. 3B, the VDW radius of sulfur, 1.85 Å, is larger than
that of oxygen, 1.40 Å (35). In addition, the thiocarbonyl bond is
0.44 Å longer than the carbonyl bond (19, 21), and S is a poor
hydrogen bond acceptor relative to O (36). These physical and chemical
characteristics of sulfur translated into an ~10° opening of the
thioG7-C20 base pair toward the major groove (Fig. 3C) with
a weakening of the hydrogen bond formed between thioG7 S-6 and
C20 H-42. Although modest, the increased distance between the heavy
atoms involved in the central hydrogen bond of the thioG7-C20 base pair
relative to the G7-C20 base pair (Fig. 3B) was consistent
with a weakening of the central hydrogen bond as well.
As the above-mentioned characteristics of S were taken into account in
the parameterization of thioG and the derivation of base pairing
constraints for the thioG7-C20 base pair, the structural effects
observed and weakened hydrogen bonds were, in fact, what would be
predicted by high level molecular modeling. These modest structural
perturbations, however, could not account for the significant differences observed in the thermal stability of the two DNA duplexes, the large chemical shift difference between the thioG7 H-1 and G7 H-1 resonance positions, and the biochemical and biological effects
of thioG-modified DNA on enzyme activities, protein recognition, and
cellular response.
ThioG Significantly Alters the Dynamics of Duplex DNA--
As
shown in Fig. 2D, the thioG7 imino resonance (circled) was
less intense than the G7 imino resonance (Fig. 2C, circled). As a consequence, cross-peak intensities involving the thioG7 imino
were also diminished (Fig. 2, D and F) relative
to cross-peak intensities involving the G7 imino (Fig. 2, C
and E). In addition, it was initially observed that, in
contrast to all other observed imino resonances including that of G7,
the intensity of the thioG7 imino resonance decreased significantly as
the mixing time was increased in two-dimensional WATERGATE
NOESY experiments (data not shown). These data suggested that the
thioG7 imino proton exchanged more rapidly with water than the G7
imino. Direct measurements showed that imino proton exchange rates of
corresponding iminos for the G-C and thioG-C duplexes were very
similar, with the exception of those for the G7 and thioG7 iminos (Fig.
4A). The G7 imino proton
exchanged more slowly (kex = 0.5 s
Duplex DNA is highly dynamic and, therefore, is continually in motion.
For example, local disruption and transient opening of individual base
pairs occur naturally on the millisecond time scale (34, 37). As base
pairs fluctuate between opened and closed states, the rate at which an
individual imino proton exchanges with solvent is governed, in part, by
the lifetime of that base pair or the amount of time spent in the
closed, base paired state (34). Once open, imino proton exchange can
occur, but among other factors, is dependent on the chemical
environment of the imino proton (i.e. neighboring S
versus O). To determine the basis for the significant
difference in exchange rates of the G7 and thioG7 imino protons with
water, imino proton exchange experiments were performed in the presence
of increasing concentrations of Tris catalyst (34). By extrapolation to
infinite catalyst concentration, the data showed that the base pair
lifetime of thioG7-C20 (8 ms) was ~80-fold shorter than the base pair
lifetime of G7-C20 (671 ms) (Fig. 4B). This means that the
thioG7-C20 base pair spent more time in the open state than the G7-C20
base pair, making thioG7 H-1 more accessible to solvent for exchange to
occur (15, 34, 37). Therefore, thioG7 H-1 exchanged more rapidly with water than G7 H-1. As for the subtle structural perturbation, the
effects of thioG on base pair dynamics were highly localized. For
example, base pair lifetimes involving adjacent residues, G6 and G11,
for both DNA duplexes were similar. (Base pair lifetimes involving T
residues more immediately on the 3' side of G7 or thioG7 than G11 could
not be determined due to resonance overlap and exchange
broadening.)
Insights into the Biochemical and Therapeutic Effects of
Thiopurines--
The present studies define for the first time the
biophysical properties of thioG-modified duplex DNA. Previous studies
of the effects of thioG on duplex DNA have been attempted using
palindromic DNA with two symmetrically related thioG-C base pairs (38). However, in this context, instability of the DNA precluded structure determination using NMR spectroscopy. Moreover, the proximity of the
two thioG sites in the palindromic DNA previously studied is probably
not representative of the frequency with which thioG sites occur in the
DNA of patients after thiopurine treatment (5, 39). These issues were
avoided here by studying DNA substituted with thioG at one central site.
In contrast to other DNA-modifying chemotherapeutic agents
(e.g. cisplatin (40)) and DNA alkylating agents (41)), which have been shown to grossly alter the structure of DNA, the present studies show that thioG, in the context of a thioG-C base pair, results
in modest, localized changes in DNA structure. These results are not
unexpected as the only difference between thioG and guanine is the
substitution of S at the O-6 position of guanine. Because dthioGTP is
so similar to the DNA precursor, dGTP, dthioGTP is readily incorporated
into duplex DNA by DNA polymerase during replication (39, 42). The
lesion is initially tolerated due to the minimal perturbation imposed
on DNA structure as reported herein.
Although highly focused as well, the effects of thioG on base pair
dynamics are much more pronounced than those on structure alone. The
thioG-C base pair is physically disrupted and transiently opens for an
extended period of time compared with normal G-C base pairs. The
magnitude and nature of these effects are consistent with the
significant decrease in the thermal stability of the thioG-C duplex
relative to the G-C duplex (31), the large upfield shift of the thioG7
H-1 resonance frequency relative to that of G7 H-1 (34) and the
dramatic increase in the exchange rate of thioG7 H-1 with water
compared with that of G7 H-1 (34). Based on these data, it is logical
to propose that increased base pair dynamics and decreased stability of
a thioG-C base pair are the mechanism by which specific DNA-processing
enzyme activities and DNA-protein interactions are altered in the
presence of substrates containing a thioG-C base pair.
For example, as previously mentioned, a multiprotein complex containing
GAPDH has recently been shown to discriminate between normal G-C and
modified thioG-C base pairs and bind DNA substrates containing the
thioG-C lesion (7). Because the structural effects of thioG on duplex
DNA are very small, discrimination is most likely due to the
significant alteration of base pair dynamics caused by thioG.
Functional groups of the thioG-C base pair, sometimes involved in W-C
hydrogen bonds, are exposed to solvent in the open conformation and
provide a unique and accessible substrate for protein binding.
RNase H removes RNA primers from Okazaki fragments during lagging
strand DNA synthesis (5, 43). Human (44), bacterial (5, 45), and
retroviral (12, 46) RNase H activities are significantly inhibited
toward DNA-RNA heteroduplex substrates containing thioG in the DNA
strand opposite C in the RNA strand. RNase H activity is specifically
inhibited in the vicinity of the thioG-C base pair (5, 12). Transient
opening of the thioG-C base pair for an extended period of time, as
described in the present work, may locally alter the width of the minor
groove, the feature of DNA-RNA heteroduplexes shown to be most
important for recognition by RNase H (43), thereby impeding RNase H
activity. Alternatively, the perturbed base pair dynamics may
sterically hinder the binding of RNase H to the heteroduplex.
Topo II induces double strand breaks (DSBs) in duplex DNA to relax the
supercoiling introduced ahead of the replication fork and transcription
machinery, in addition to its roles in other processes of DNA
metabolism (47). Compared with unmodified substrates, Topo II-induced
DSBs have been shown to be increased in duplex DNA containing a thioG-C
base pair in the Topo II cleavage site (13). These effects are
consistent with inhibition of the religation step involved in Topo
II-mediated DSBs (13). Interestingly, T4 DNA ligase activity toward
duplex DNA containing a thioG-C base pair adjacent to the ligation site
has also been shown to be inhibited relative to the activity toward
unmodified substrate (8). Together, these data suggest that it is the
inhibition of DNA ligase activity, not an actual increase in Topo II
cleavage activity, that accounts for the observed increase of Topo
II-mediated DSBs. The presence of a thioG-C base pair near a DSB,
through its effects of base pair dynamics, may locally destabilize
duplex DNA so as to inhibit the ligation reactions catalyzed by Topo II
and DNA ligase and the nuclease reaction catalyzed by RNase H.
While inhibitors of DNA synthesis protect cells from the cytotoxic
effects of thiopurines (48), dthioGTP is readily incorporated into DNA
(39, 42) without immediate deleterious effects on DNA synthesis or cell
viability (5, 6, 48, 49). Rather, the cytotoxicity observed in response
to thiopurines is a delayed effect associated also with the delayed
arrest of the cell division cycle at S and G2 phase (5, 6).
These cellular responses underlie the antileukemic effects of
thiopurines. The delayed nature of these responses indicates that
subsequent rounds of DNA replication, in which thioG is in the template
strand, are impeded resulting in cytotoxicity. In vitro
studies show that thioG-C versus thioG-T is 300-fold more
common in MP-treated cells and is thus the more therapeutically
relevant thioG-containing base pair to consider in this context (7).
The present studies, therefore, define the nature of perturbations
caused by thioG-C incorporation into DNA that provide the basis for
understanding the antileukemic effects of thiopurines. These same
perturbations provide a mechanism by which specific DNA-processing
enzyme activities and DNA-protein interactions are altered in the
presence of substrates containing a thioG-C base pair.
It is interesting to note that both inhibition of RNase H activity when
thioG is positioned in the DNA strand and an increase in irreparable
Topo II-mediated DSBs induced by thioG ahead of the replication fork or
transcription machinery are effects that, under physiological
conditions, would be observed when thioG is necessarily in the template
strand during subsequent rounds of replication. These observations
implicate RNase H and Topo II complexes as playing direct roles in
mediating the delayed antileukemic effects of thiopurines.
In summary, due to its biomolecular compatibility, thioG is metabolized
and ultimately incorporated into duplex DNA by DNA polymerase during
replication. Other DNA processing enzymes (i.e. RNase H,
Topo II, and DNA ligase), however, are not immune to the
physical-chemical effects of thioG. These enzymes are exquisitely sensitive to the presence of thioG in DNA strands, and we attribute this to the striking and localized effects of thioG on base pair stability and dynamics. This mechanism of action is particularly insidious in that thioG escapes detection and persists during one round
of replication, only to arrest DNA replication during the next round
through its effects on the aforementioned enzymes. It is also possible
that the specific binding of the GAPDH-containing protein complex to
thioG sites contributes to the lethality of thioG incorporation by
triggering apoptosis through a currently unknown mechanism. Thus, our
structural and dynamic results for thioG-modified duplex DNA offer
unique insights into its dichotomous mechanism of action, being
structurally silent on the one hand and producing lethal effects on
base pair stability and dynamics on the other. This mechanism is in
striking contrast to the gross effects of, for example, cisplatin and
alkylating agents on DNA structure and function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
ThioG decreases the thermal stability of
duplex DNA and has a large but localized effect on the chemical
properties of duplex DNA. A, sequence of thioG-C and
G-C DNA duplexes, where X = deoxythioguanosine and
deoxyguanosine, respectively. B, thermal denaturation curves
for G-C (filled circles) and thioG-C (open
squares) DNA duplexes. The horizontal line intersects each of the
curves at the melting temperature (Tm) defined
as the temperature at which half of each duplex exists in the denatured
single strand state. C, proton chemical shift differences
between G-C and thioG-C DNA illustrated on a per residue basis.
D, proton chemical shift differences for individual protons
in residues 7 and 20 between G-C and thioG-C DNA. The C20 H-42
proton is underlined to distinguish it as the amino proton on N-4
involved in hydrogen bonding.
3 mM) in Buffer A was
monitored at 260 nm (Hewlett-Packard Vectra XA UV-visible spectrophotometer) over a temperature range of 10-70 °C. Absorbance was measured every 0.5 °C as the temperature was increased at a rate
of 0.5 °C min
1 (1-min equilibration period between
measurements). Melting temperatures (Tm),
corresponding to the midpoints of the thermal denaturation curves, were
obtained by the analysis of normalized absorbance data using the
manufacturer's software.
dihedral angle (C-4'-C-3'-O-P) was
broadly constrained to 200 ° ± 60 ° (18).
1, cooling to 300 K at 25 K
sec
1 and equilibrating at 293 K over 250 ps. Ensembles of
structures were chosen based on R-factor and consisted of 20 structures
each. Structural analysis was performed using the 3DNA program
(27).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 2.
ThioG exists in the keto form in
duplex DNA. A, tautomeric forms of thioG. The imino
proton (circled) at the N-1 position of the keto form of
thioG is chemically distinct from the sulfhydryl proton
(boxed) at the S-6 position on the enol form. B,
schematic diagram of protons near the imino proton of G7 that should
give rise to NOESY cross-peaks. C and D, excerpts
from 100 ms 600 MHz two-dimensional WATERGATE NOESY spectra for G-C
(C) and thioG-C (D) DNA showing sequential
cross-peaks for G7 H-1 (or thioG7 H-1) to G6 H-1 and T19 H-3.
The diagonal peaks for the imino protons for position 7 are
circled. Similar data are shown for T17 H-3 for reference.
E and F, excerpts from 200 ms 600 MHz
two-dimensional WATERGATE NOESY spectra for G-C (E) and
thioG-C (F) DNA showing cross-peaks for G7 H-1
(E) and thioG7 H-1 (F) to neighboring aromatic
protons. The C20 H-42 proton is underlined to distinguish it
as the amino proton on N-4 involved in hydrogen bonding.
Data collection and structural statistics of G-C and thioG-C ensembles
1 with standard
deviations in parentheses) for each ensemble are reported as mean
values over those 20 structures. rmsd values were calculated over heavy
atoms for structures in which terminal residues (1, 2, 12, 13, 14, 15, 25, and 26) were not considered. The distance energy corresponds to
constraints applied to distances between heavy atoms involved in base
pair hydrogen bonds and distances between exchangeable protons in base
pairs. The relaxation energy corresponds to NOE volume constraints.
View larger version (32K):
[in a new window]
Fig. 3.
The effects of thioG on duplex DNA
structure are subtle and localized to the base pair containing
thioG. A, views of the ensembles of G-C and thioG-C DNA
structures. Each ensemble consists of 20 structures. The dotted
boxes encompass the G7-C20 and thioG7-C20 base pairs of the G-C
and thioG-C duplexes, respectively. B, CPK drawings
of a G7-C20 and thioG7-C20 base pair from each of the respective
ensembles. Distances between heavy atoms involved in hydrogen bonds are
reported. C, opening of each of the interior base pairs of
the G-C (black box) and thioG-C DNA (white box)
structures. A positive angle corresponds to opening toward the major
groove, whereas a negative angle corresponds to opening toward the
minor groove.
1) than other G-C imino protons, as expected, due to its
central location and involvement in a base pair with three hydrogen
bonds (37). Although the thioG7 imino proton was also centrally
located, it exchanged at a rate (kex = 25.0 s
1) much greater than that of the G7 imino proton.
View larger version (14K):
[in a new window]
Fig. 4.
The imino proton of thioG7 exchanges faster
than that of G7, and the base pair lifetime of thioG7-C20 is
~80-fold shorter than that of G7-C20.
A, imino proton exchange rates for all interior residues of
the G-C (black box) and thioG-C (white box) DNA
duplexes. B, imino proton exchange times (inverse of
exchange rate) versus [Tris catalyst] 1 for
G7 (filled) and thioG7 (open). Lines representing the best
linear fit to the data are drawn (kaleidograph). Exchange times at
infinite catalyst concentration (y-intercept) were
interpreted as the base pair lifetimes of G7-C20 and thioG7-C20.
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ACKNOWLEDGEMENTS |
---|
We thank the anonymous reviewers for constructive criticism and insightful suggestions on an earlier draft of the manuscript. We also thank David Live and Ian Armitage (University of Minnesota) as well as Ron Venters and Len Spicer (Duke University) for help acquiring 800 MHz NMR spectra, Charles Ross for assistance with NMR and structure calculation software and data manipulation, and David Langley (Bristol Myers-Squibb) for modifying the Amber98 force field for compatibility with XPLOR.
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FOOTNOTES |
---|
* 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.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M204243200
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ABBREVIATIONS |
---|
The abbreviations used are: MP, mercaptopurine; ALL, acute lymphoblast leukemia; dthioGTP, deoxy-6-thioguanosine 5'-triphosphate; thioG, thioguanine; MMR, mismatch repair; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; NOE, Nuclear Overhauser effect; DQF, double quantum-filtered; VDW, Van der Waals; rMD, restrained molecular dynamics; B-DNA, B-form DNA; W-C, Watson-Crick; DSBs, double strand breaks.
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