Structure and Dynamics of Thioguanine-modified Duplex DNA*,

Lilla SomervilleDagger §, Eugene Y. KrynetskiDagger §, Natalia F. KrynetskaiaDagger §, Richard D. Beger, Weixing Zhang||, Craig A. Marhefka**, William E. EvansDagger §DaggerDagger, and Richard W. Kriwacki||§§¶¶

From the Departments of Dagger  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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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.

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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.

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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. 


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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.

Thermal Denaturation Studies-- The absorbance of each duplex DNA (2.3 × 10-3 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.

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 epsilon  dihedral angle (C-4'-C-3'-O-P) was broadly constrained to 200 ° ± 60 ° (18).

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-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).

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.

    RESULTS AND DISCUSSION
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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.


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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.

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.

                              
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Table I
Data collection and structural statistics of G-C and thioG-C ensembles
Each ensemble consists of 20 structures. The R-factor, root mean square deviation (rmsd) and energies (kcal mol-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.


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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.

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-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.


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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.

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.

    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.

    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/).

Dagger Dagger 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

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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