Structure of an Oligodeoxynucleotide Containing a 1, N 2

U. S., Moe, J. G., Reddy, G. R., Weisenseel, J. P., Marnett, L. J., and Stone, ... Hai Huang , Hao Wang , R. Stephen Lloyd , Carmelo J. Rizzo and ...
23 downloads 0 Views 549KB Size
Download PDF
Chem. Res. Toxicol. 2002, 15, 127-139

127

Structure of an Oligodeoxynucleotide Containing a 1,N2-Propanodeoxyguanosine Adduct Positioned in a Palindrome Derived from the Salmonella typhimurium hisD3052 Gene: Hoogsteen Pairing at pH 5.2 Jason P. Weisenseel,† G. Ramachandra Reddy,‡ Lawrence J. Marnett,‡ and Michael P. Stone*,† Departments of ⊥Chemistry and #Biochemistry, Center in Molecular Toxicology, A.B. Hancock, Jr., Memorial Laboratory for Cancer Research, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37235 Received July 2, 2001

The structure of the 1,N2-Propanodeoxyguanosine (PdG) adduct was determined at pH 5.2 in the oligodeoxynucleotide duplex 5′-d(CGCGGTXTCCGCG)3′‚5′-d(CGCGGACACCGCG)-3′ (X ) PdG). This sequence, referred to as the -TXT- sequence, is contained within the Salmonella typhimurium hisD3052 gene and contains a palindrome, representing a potential hotspot for frameshift mutagenesis. PdG provides a model for the primary adduct induced in DNA by malondialdehyde, the 3-(2′-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-a]-purin-10(3H)-one (M1G) lesion. The solution structure was refined by molecular dynamics calculations restrained by a combination of NMR-derived distances and dihedral angles, using a simulated annealing protocol. PdG introduced a localized perturbation into the sequence at base pair X7‚C20, which was pH-dependent. At neutral pH, conformational exchange resulted in spectral line broadening, and it was not possible to determine the structure. A stable structure was observed at pH 5.2 in which PdG rotated about the glycosyl bond into the syn conformation. This placed the exocyclic moiety into the major groove of the duplex. PdG formed a protonated Hoogsteen pair with nucleotide C20 in the complementary strand. The pseudorotation of the deoxyribose at C20 was altered to an approximately equal blend of C2′-endo and C3′-endo structures. However, these made little difference in the overall structure of the modified oligodeoxynucleotide. The structure was compared to that of PdG in the 5′-d(CGCXCGGCATG)-3′‚5′-(CATGCCGCGCG)3′ sequence (the -CXC- sequence) at pH 5.8 [Singh, U. S., Moe, J. G., Reddy, G. R., Weisenseel, J. P., Marnett, L. J., and Stone, M. P. (1993) Chem. Res. Toxicol. 6, 825-836]. A sequence effect was observed. When PdG was placed into the -TXT- sequence at low pH, the structural perturbation was limited to the X7‚C20 base pair. In contrast, when PdG was placed into the -CXC- sequence at low pH, both the modified base pair and its 3′-neighbor base pair were disrupted. The results are discussed in the context of differential outcomes for site-specific mutagenesis and replication bypass experiments when PdG was placed in the -TXT- and -CXCsequences, respectively.

Introduction Exocyclic adducts arise in DNA from endogenous exposure to lipid peroxidation products, such as malondialdehyde (MDA)1 (for a review, see refs 1-3). MDA reacts with DNA as a bis-electrophile to form the exocyclic guanine adduct M1G1 [3-(2′-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-a]-purin-10(3H)-one] (4-8). M1G also arises as a consequence of DNA oxidative damage, resulting in the formation of base propenals that can

transfer their oxopropenyl group to deoxyguanosine (9) (Scheme 1). The base propenals represent a substantial source of the M1G lesion (10). M1G has been observed in DNA from rodent (11) and human (12, 13) tissue samples, as have other exocyclic purine lesions (14-16), suggesting their formation in vivo. M1G was quantitated by mass spectroscopic (17, 18), postlabeling (19, 20), and immunochemical (21) techniques. It is an abundant adduct in human DNA (18-20, 22).

* To whom correspondence should be addressed. Phone: (615) 3222589. Fax: (615) 343-1234. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Biochemistry. 1 Abbreviations: COSY, correlation spectroscopy; DQF-COSY, doublequantum-filtered correlation spectroscopy; M1G, 3-(2′-deoxy-β-D-erythropentofuranosyl)pyrimido[1,2-a]-purin-10(3H)-one; MDA, malondialdehyde; NOESY, two-dimensional NOE spectroscopy; OPG, N2-(3-oxo1-propenyl)-dG; PdG, 1,N2-propanodeoxyguanosine; PEM, potential energy minimization; rMD, restrained molecular dynamics; rmsd, rootmean-square-deviation; R1x, sixth root factor residual; TPPI, timeproportional phase increment; τc, correlation time.

M1G is stable in nucleotides and single-stranded DNA at neutral pH. Under basic conditions, it converts to its N2-(3-oxo-1-propenyl)-dG (OPG) derivative. When M1G is placed at neutral pH into DNA opposite deoxycytosine, a rapid, spontaneous, and quantitative conversion to the OPG derivative is facilitated. Upon denaturation of the duplex, M1G is regenerated (Scheme 1). Ring-opening does not occur at neutral pH in DNA if thymine is placed opposite to M1G. These observations led to the conclusion that cytosine in duplex DNA catalyzes the transformation

10.1021/tx0101090 CCC: $22.00 © 2002 American Chemical Society Published on Web 01/24/2002

128

Chem. Res. Toxicol., Vol. 15, No. 2, 2002

Weisenseel et al.

Scheme 1. (A) Formation of M1G from Malondialdehyde or from Base Propenals; Thymine Propenal Is Shown as a Representative Propenal;a (B) M1G Is Stable in Single-Stranded DNAb

a Note the numbering scheme for M G in which the imidizole proton is H2, corresponding to the H8 proton in purines. b In duplex 1 DNA, when placed opposite deoxycytosine, it is spontaneously and quanitatively converted to N2-(3-oxo-1-propenyl)-dG, the OPG derivative

of M1G to its ring-opened OPG derivative, probably by a mechanism involving the exocyclic amino group of the complementary cytosine (23). The major deoxyguanosine adduct derived from acrolein, γ-hydroxyl-1,N2-propano2′-deoxyguanosine, also exists opposite deoxycytosine in duplex DNA primarily as its ring-opened derivative (24). Malondialdehyde is a frameshift mutagen in the hisD3052 tester strain of Salmonella typhimurium (25). The mutable region of the hisD3052 gene contains several sequence motifs associated with frameshift mutagenesis. One such sequence is 5′-d(ATCGCGCGGCATG)-3′. It contains an iterated repeat of CG dinucleotide units (26). Another is 5′-d(CGCGGTGTCCGCG)-3′, in which the central -TGT- sequence is flanked on the 5′ side by 5′-CGCGG-3′ and on the 3′-side by 5′-CCGCG-3′ sequences, to form a palindrome. Site-specific mutagenesis experiments resulted in M1G f A transitions and M1G f T transversions, but not frameshifts, when 5′-d(GGTXTCCG)-3′ (X ) M1G) was inserted into a viral replication vector and replicated in Escherichia coli (27). These studies verified that M1G is a mutagenic lesion in the bacterial system. The spontaneous ring opening to the OPG derivative precludes structural examination of intact M1G placed opposite dC in duplex DNA. However, the structure of M1G in the d(ATCGCXCGGCATG)-3′ (X ) M1G) sequence, when placed opposite a two base deletion in the complementary strand, was determined. M1G was in the anti conformation about the glycosyl bond and inserted into the duplex. The 3′-neighboring cytosine was extruded toward the major groove (28). The structure of OPG in d(ATCGCXCGGCATG)‚d(CATGCCGCGCGAT), X ) OPG, was determined (29). OPG maintained stacking interactions with neighboring bases. It was not Watson-Crick

hydrogen bonded. The cytosine complementary to OPG was pushed toward the major groove but maintained partial stacking with neighboring bases. The modified guanine remained in the anti conformation, while the OPG propenyl moiety was in the minor groove. The 1,N2-propanodeoxyguanosine adduct (PdG) (30) provides a stable structural model for M1G. It differs significantly from M1G in that it is chemically stable and does not undergo ring-opening to OPG. This chemical difference can be exploited to determine how exocyclic 1,N2-dG lesions are accommodated by the DNA duplex under conditions in which M1G spontaneously undergoes ring-opening to OPG when placed opposite dC. Such information may provide insight as to why the OPG lesion is more stable than M1G in duplex DNA. The PdG lesion also may provide insight into how M1G may be accommodated by a replication intermediate. Several site-specific mutagenesis studies utilizing PdG have been reported (31-33). Its mutagenicity was greater in E. coli than in simian kidney (COS) cells (33), suggesting differences in bacterial vs mammalian biological processing of the lesion. As observed for M1G, both PdG f A transitions and PdG f T transversions occurred when 5′-d(CGCGGTXTCCGCG)-3′ (X ) PdG) was inserted into a viral replication vector and replicated in E. coli (31, 34). PdG induced equal numbers of PdG f A transitions and PdG f T transversions, although the total mutation frequency was ∼2-fold greater than with M1G. Base pair substitutions were not observed when PdG was placed in 5′-d(ATCGCXCGGCATG)-3′ (35). Both M1G and PdG were substrates for repair in E. coli by the nucleotide excision repair complex (36, 37). PdG blocked replication of synthetic template-primers by polymerases in vitro (32). The replication bypass of PdG in vitro

Structure of PdG in the -TXT- Sequence

depended upon the identity of the polymerase attempting to replicate past the lesion, and was also sequence dependent (38, 39). Previous structural studies revealed that when mispaired with dA, PdG exhibited X (syn)‚A(anti) pairing at pH 5.8, and simultaneous partial intercalation of the complementary X and A bases at pH 8.9. When mispaired with dG, PdG exhibited X(syn)‚G(anti) pairing which was pH-independent (40-43). The exocyclic ring of PdG was inserted into the DNA duplex when positioned opposite an abasic site (44). When placed in 5′- d(CGCXCGGCATG)-3′ at pH 5.8, PdG induced a localized structural perturbation involving the modified base pair and its 3′neighbor. PdG rotated about the glycosyl bond into the syn conformation, and the 3′-neighbor base pair existed in a mixture of Watson-Crick and Hoogsteen conformations (45). Subsequently, when PdG was placed in 5′d(ATCGCXCGGCATG)-3′ opposite a 2 base deletion in the complementary strand, PdG was in the anti conformation about the glycosyl bond and inserted into the DNA duplex. The 3′-neighbor dC was extruded toward the major groove (46, 47). PdG reduced the thermal stability, transition enthalpy, and transition free energy of duplex DNA when positioned opposite cytosine or adenine, and the destabilization of the duplex was not sensitive to whether the base opposite the lesion was adenine or cytosine (48). The present work examines PdG in 5′-d(CGCGGTXTCCGCG)3′‚5′-d(CGCGGACACCGCG)-3′ (X ) PdG), at low pH. This is named the -TXT- duplex. The data show that PdG introduces a localized perturbation at base pair X7‚C20, which is pH-dependent. At physiological pH, the X7‚C20 base pair exists as an equilibrium blend of conformations that differ in their protonation state. The low pH structure predominates at pH 5.2, thus allowing its structure to be established. At pH 5.2, PdG rotates about the glycosyl bond into the syn conformation, thus placing the exocyclic moiety into the major groove. In this conformation, PdG forms a protonated Hoogsteen pair with nucleotide C20 in the complementary strand. The pseudorotation of the deoxyribose unit at the complementary base C20 is altered to an approximately equal blend of C2′-endo and C3′-endo conformers, which has little effect on the overall duplex structure. The structure of PdG in the -TXT- oligodeoxynucleotide at acidic pH differs from that of PdG in 5′-d(CGCXCGGCATG)-3′-5′(CATGCCGCGCG)-3′ (the -CXC- sequence) at acidic pH, in which PdG was placed within a CG iterated repeat (45). In the -CXC- sequence, PdG also formed a protonated Hoogsteen pair with the complementary cytosine, but in that sequence, the 3′-neighbor base pair also equilibrated between Hoogsteen and Watson-Crick base pairing. The structural differences parallel differences observed for site-specific mutagenesis and replication bypass experiments when PdG was inserted into the -TXT- and -CXC- sequences, respectively.

Materials and Methods Materials. Oligodeoxynucleotides were synthesized and purified by anion-exchange chromatography by the Midland Certified Reagent Co. (Midland, TX). The 5′-dimethoxytrityl phosphoramidite of PdG was synthesized and purified (30). It was incorporated into 5′-d(CGCGGTXTCCGCG)-3′ (X ) PdG) and purified by anion-exchange chromatography by the Midland Certified Reagent Co. The oligodeoxynucleotides were identified by MALDI mass spectrometry. Purity was assessed by capillary

Chem. Res. Toxicol., Vol. 15, No. 2, 2002 129 gel electrophoresis. Oligodeoxynucleotides were desalted either by chromatography on Sephadex G-25 (Amersham Pharmacia Inc., Piscataway, NJ) or dialysis (Spectrum, Houston, TX). Oligodeoxynucleotide concentrations were determined by UV absorption at 260 nm. The calculated extinction coefficient, in which it was assumed that dG ) PdG, was 1.26 × 105 M-1 cm-1 for the modified strand 5′-d(CGCGGTXTCCGCG)-3′. The validity of this assumption for PdG was not checked, except to the extent that the utilization of this calculated extinction coefficient allowed successful preparation of duplexes in of approximate 1:1 stoichiometry, which were subject to subsequent purification using hydroxylapatite. The calculated extinction coefficient was 1.12 × 105 M-1 cm-1 for the complementary strand 5′-d(CGCGGACACCGCG)-3′ (49). Annealing the duplexes presented problems due to the high thermal stability of hairpins formed by the modified and complimentary strands (50). The strands were annealed at a concentration of 9.0 mM in 10 mM NaH2PO4, 0.1 M NaCl, and 50 µM EDTA (pH 7.0), to maximize duplex formation. The solutions were heated to 95 °C for 5 min and cooled to room temperature. The duplexes were eluted from DNA Grade hydroxylapatite with a gradient from 10 to 200 mM NaH2PO4 in 0.1 M NaCl, 50 µM EDTA (pH 7.0). Duplexes were desalted with Sephadex G-25. UV Melting. Experiments were carried out on a Cary 4E spectrophotometer (Varian Associates, Palo Alto, CA) with 8-9 µM strand concentration in 1 mL of 10 mM NaH2PO4, 1.0 M NaCl, and 50 µM Na2EDTA (pH 7.0) in a 1.0 cm cuvette. Experiments were also conducted at as a function of pH and NaCl concentration. Samples were heated at a rate of 1 °C/min and data were collected at a rate of 1 point/min. NMR. Samples were at 3.6 mM strand concentration. Samples for the observation of nonexchangeable protons were dissolved in 500 µL of 10 mM NaH2PO4, 100 mM NaCl, 50 µM Na2EDTA (pH 5.2). They were exchanged with D2O and suspended in 500 µL of 99.996% D2O. Samples for the observation of exchangeable protons were dissolved in 500 µL of 1 mM NaH2PO4, 100 mM NaCl, and 50 µM Na2EDTA (pH 5.2) containing 9:1 H2O:D2O. The temperature was 20 °C for observation of nonexchangeable protons and 5 °C for observation of exchangeable protons. Chemical shifts were referenced to water. Data were processed using FELIX 97.0 (Accelrys Inc., San Diego, CA) on Silicon Graphics workstations (Silicon Graphics, Inc., Mountain View, CA). For assignment of exchangeable protons, NOESY experiments used the watergate pulse sequence (51). The mixing time was 250 ms. For assignment of nonexchangeable protons and the derivation of distance restraints, NOESY experiments used TPPI quadrature detection and a mixing time of 250 ms. A relaxation delay of 2 s was used. Experiments were recorded with 512 real data in the t1 dimension and 2048 real data in the t1 dimension. Data in the t1 dimension were zero-filled during processing to create a matrix of 2048 × 2048 real points. A skewed sinebell-square apodization with an 80° phase shift and a skew factor of 0.7 was used in both dimensions. DQFCOSY experiments were measured with TPPI quadrature detection and presaturation of the residual water during the relaxation delay. Spectra were measured with 1024 real data in the t1 dimension and 2048 real data in the t2 dimension. The data in t1 were zero-filled during processing to create a matrix of 2048 × 2048 real points. A skewed sinebell-square apodization with a 60° phase shift and a skew factor of 0.62 was used in both dimensions. 1H-31P correlation experiments (52) using selective IBURP shaped pulses (53) were collected with 256 real points in the 1H dimension over a 1250 Hz spectral width and 256 real points in the 31P dimension over a 405 Hz spectral width. Data were processed by zero-filling both dimensions, to obtain 512 × 512 matrixes. A sinebell square apodization with a 72° phase shift was used in the 1H dimension and Gaussian multiplication function in the 31P dimension, with a line broadening of -9.0 and 0.2 Gaussian broadening. Chemical shifts were referenced externally to trimethyl phosphate. Distance Restraints. NOE-derived distances from crosspeak volumes measured at a mixing time of 250 ms were

130

Chem. Res. Toxicol., Vol. 15, No. 2, 2002

calculated using MARDIGRAS v3.2 (54, 55). Cross-peaks from resonances that exhibited chemical shift degeneracy required special treatment. Volumes integrated from these were divided in half and assigned to the two symmetry-related nucleotides. Isotropic correlation times of 2, 3, and 4 ns were used. The volume error was one-half the volume of the smallest peak. The RANDMARDI algorithm carried out 50 iterations for each set of data, randomizing peak volumes within limits specified by the input noise level (56). The distances were averaged. The standard deviation in a particular distance served as the error bound for the distance. Distance restraints were divided into classes weighted according to the error assessed in their measurement. Class 1, class 2, and class 3 distances were calculated from completely resolved, slightly overlapped, or medially overlapped cross-peaks, respectively, that were at least 0.5 ppm away from the water resonance or the diagonal line of the spectrum. Class 4 distances were calculated from all other cross-peaks. Empirical restraints preserved Watson-Crick hydrogen bonding and prevented propeller twisting between base pairs (41). NOEs that did not have a distance calculated by MARDIGRAS were assessed as strong, medium, or weak. These were assigned as 1.8-2.8, 1.8-3.8, or 1.8-5.0 Å, respectively. Negative restraints prevented protons for which no NOE was observed from moving closer than 5 Å. Dihedral Angle Restraints. Deoxyribose conformation was graphically determined using sums of 3J 1H coupling constants from DQF-COSY data (57). These were fit to curves relating coupling constant to the pseudorotation angle (P), sugar pucker amplitude (Φ), and the percentage S-type conformation. The pseudorotation angle and amplitude ranges were converted to the dihedral angles ν0 to ν4. The 31P-H3′ 3J coupling was applied to the Karplus relationship (58) to determine the phosphodiester backbone angle , related to the H3′-C3′-O3′-31P angle by a 120° shift. The phosphodiester angle ζ was calculated from the correlation between  and ζ in B-form DNA (59). The ζ restraints were not applied at and adjacent to PdG. If the Σ H4′ J coupling was less than 11 Hz, the phosphodiester angle γ was concluded to be in the range 10-110° (60). If the cross-peak between the aromatic H8/H6 and the H5′/H5′′ protons had a smaller volume than the cross-peak between the aromatic H8/H6 and the H1′ protons, it was concluded that the distance between the aromatic H8/H6 and H5′/H5′′ protons was >3.8 Å. If the cross-peak between the H2′ and H5′/H5′′ protons had smaller volume than the cross-peak between the H2′ and H1′ protons, it was concluded that the distance between the H2′ and H5′/H5′′ protons was >3.0 Å. When both of these conditions were met, γ was concluded to be 60 ( 40°. Consequently, H5′ and H5′′ faced the exterior of the helix and the H3′ to H5′′ distance was shorter than the H3′ to H5′ distance. Stereospecific assignment of the H5′ and H5′′ protons was then made by comparing the volumes of the H3′ to H5′ and H5′′ NOE peaks. Restrained Molecular Dynamics. The -TXT- oligodeoxynucleotide was constructed in both A-DNA and B-DNA forms (61) by bonding a propano group to N1 and N2 of G7 using Insight II (Accelrys Inc., San Diego, CA). Partial charges on PdG were approximated by SCF calculations using a neutral total charge, utilizing the MNDO method and the program MOPAC (62) (Supporting Information). The duplexes were energy minimized by conjugate gradients for 200 iterations without experimental restraints to give starting structures TXT-IniA and TXTIniB. Calculations in vacuo without explicit counterions utilized X-PLOR (63) and the CHARMM (64) force field. The electrostatic term used the Coulomb function based on a reduced charge set of partial charges and a distance dependent dielectric constant of 4.0. The van der Waals term was approximated using the Lennard-Jones potential energy function. The cutoff radius for nonbonding interactions was 11 Å. The nonbonded pair list was updated if any atom moved more than 0.5 Å. The restraint energy function contained terms describing distance and dihedral restraints, both in the form of square well potentials (65). Bond lengths involving hydrogens were fixed with the SHAKE algorithm (66).

Weisenseel et al. A 40 ps simulated annealing protocol used an integrator time step of 1 fs. Ten structures were calculated from each starting structure. Random velocities were assigned, fitting a MaxwellBoltzmann distribution for 2000 K, and coupled to a temperature bath with a constant of 0.05 ps (67). An initial hightemperature period of 20 ps at 2000 K was followed by 5 ps of cooling and an equilibration period of 15 ps at 300 K. An initial force constant of 50.0 kcal mol-1 Å-1 was used for class 1 restraints. The force constants for class 2, 3, and 4 restraints were set to 80, 60, and 40%, respectively, of the value for class 1. The initial value of the force constant for the base pairing restraints was set to 0.0 kcal mol-1 Å-1. Force constants were maintained at the initial value for the first 5 ps of the calculations. Class 1 force constants were increased to 250 kcal mol-1 Å-1 and base pairing force constants were increased to 150 kcal mol-1 Å-1 over the next 5 ps. They were maintained for the subsequent 17 ps. Force constants were scaled down to 70 kcal mol-1 Å-1 and 50 kcal mol-1 Å-1 for class 1 and base pairing restraints, respectively, over the next 3 ps and remained at these values for the final 10 ps. Structure coordinates were archived every 0.1 ps over the final 10 ps. Structure coordinates extracted from the final 4 ps were averaged and energy minimized for 200 iterations using conjugate gradients. Convergence was assessed for structures having the lowest number of deviations from the experimental distance and dihedral restraints, lowest van der Waals energy, and the lowest overall energy. CORMA Calculations and Helical Analysis. Calculation of NOE intensities from the structures emergent from rMD calculations utilized CORMA (68). Input volumes (intensities) were normalized from the intensities of protons with fixed internuclear distances (i.e., cytosine H5-H6 distance). Random noise was added to all intensities to simulate spectral noise. An isotropic correlation time (τc) of 3 ns was used. The rotation of thymidine CH3 groups was modeled using an 18-jump site model (69). A sixth root residual (R1x) factor was calculated for each structure (70). Helicoidal analysis was performed using Dials and Windows 1.0 (71).

Results Thermal Stability. PdG destabilized the -TXT- oligodeoxynucleotide duplex. The melting point of the -TXTduplex was 35 °C in 1 mM NaCl at pH 7.0. The corresponding Tm for the unmodified oligodeoxynucleotide under the same conditions was 49 °C. Thus, PdG induced a 14 °C drop in Tm. The low salt concentration was utilized, because at 1 M NaCl, the melting curve was complex due to hairpin formation as the duplex melted. The Tm was measured as a function of pH in phosphate buffer containing 1 M NaCl. The Tm increased at low pH yet remained constant at neutral pH and above. This was consistent with the notion that a more stable species was formed at lower pH. The melting points for pH 5.8, 7.0, and 8.2 samples were 41 °C ((0.3), 38 °C ((0.3), and 38 °C ((0.3), respectively. NMR experiments corroborated this finding. At pH 7, resonances for protons in both strands of the duplex at and adjacent to X7 were broadened. Many were not observable at 5 °C, suggesting disorder at the adduct site. As pH was lowered to 5.2, the resonances sharpened, suggesting a more ordered protonated structure. Accordingly, NMR structural studies were conducted at pH 5.2. Resonance Assignments. (a) Nonexchangeable Protons. An unusual feature of the NMR spectra was the chemical shift degeneracy for base pairs C1‚G26 and G13‚ C14, and G2‚C25 and C12‚G15 (Figure 1). These were the terminal and penultimate base pairs at each end of the duplex. This arose as a consequence of the palindrome about the central -TXT- sequence (Scheme 2). Sequential

Structure of PdG in the -TXT- Sequence

Figure 1. 1H NOESY spectrum showing sequential NOE connectivities for the modified and complementary strands of the -TXT- oligodeoxynucleotide. (Top) The PdG-modified strand. An interruption is observed at T6, where the T6 H1′ f X7 H2 connectivity is missing. The X7 H2 f X7 H1′ NOE is unusually large. The connectivities between X7 H1′ and G13 are uninterrupted. (Bottom) The complementary strand. No interruptions were observed in the sequential connectivities. The boxes enclosing base pairs C1‚G26, G2‚C25, C12‚G15, and G13‚C14 in the oligodeoxynucleotide sequence at the bottom of the panel indicate the chemical shift degeneracy observed for these symmetry related base pairs.

Scheme 2. PdG Hairpin Oligonucleotide Numbering Scheme and the Structure and Numbering Scheme for PdGa

a Note the imidizole proton in PdG is also numbered H2, corresponding to the H8 proton in guanine.

NOESY connectivities (72) were followed at pH 5.2 through the modified strand from nucleotides C1 to T6. The usual B-form DNA NOE between T6 H1′ and X7 H2 was missing. The intensity of the X7 H2 f X7 H1′ crosspeak was exceptionally large (Figure 2). The NOESY connectivities were uninterrupted from X7 H1′ to G13. The NOE connectivities were continuous in the complementary strand. The normally observed NOE between T6 H2′/ H2′′ and X7 H2 was also missing. The 1H assignments are provided in the Supporting Information.

Chem. Res. Toxicol., Vol. 15, No. 2, 2002 131

(b) Exchangeable Protons. Figure 3 shows the imino region of the 1H NOESY spectrum. The expected pattern of NOE cross-peaks was observed for base pairs G4‚C23, G5‚C22, and T6‚A21, which were located on the 5′ side of PdG. Likewise, NOE connectivities were observed between base pairs T8‚A19, C9‚G18, and C10‚G17 on the 3′side of PdG. The observation of these resonances suggested that Watson-Crick base pairing was intact at both base pairs T6‚A21 and T8‚A19 that flanked the PdGmodified base pair. The X7 H5 amino proton of PdG was not observed, suggesting rapid exchange with solvent. The observation at δ 9.77 ppm and δ 8.78 ppm of the C20 amino proton resonances, the nucleotide complementary to PdG, was striking (Figure 4). This represented a significant downfield shift for the cytosine amino protons at C20. (c) Deoxyribose and Backbone Angle Conformations. At the modified base pair X7‚C20, the pseudorotation of the C20 deoxyribose was altered. The ratio of S-type to N-type conformer at C20 was approximately 1:1. All other sugars for which pseudorotation could be measured were S-type. The pseudorotations of the G2, T8, G11, G15, A19, and G24 deoxyribose rings could not be determined. Few 31P resonances could be assigned due to the small distribution in their chemical shifts. The coupling constants measured from the cross-peaks, when possible, were applied to the Karplus relationship (58) to determine the dihedral angle . These fell in the range of 5-8 Hz, and the chemical shifts were consistent with a trans conformation of the backbone angle  (C4′-C3′-O3′-P). The γ backbone angles for all nucleotides were determined to fall in the range of 60 ( 40°. (d) PdG Exocyclic Ring Protons. Figure 5 shows the assignment of the PdG ring protons and also shows NOEs between these protons and DNA. The H6a,b resonances of the PdG exocyclic ring were degenerate and observed at δ 3.43 ppm. The H7a resonance was at δ 1.93 ppm, while the H7b resonance was at δ 2.04 ppm. The H8a resonance was at δ 3.56 ppm and the H8b resonance was at δ 3.87 ppm. The chemical shifts of the PdG protons in the present study approximated those of PdG embedded into the iterated (CG)3 repeat sequence (45) in which PdG was in the syn conformation exposing the exocyclic ring protons to solvent (Figure 6). A number of NOEs were observed between PdG ring protons and DNA, which served to position PdG with respect to the duplex. Most involved T6, on the 5′ neighbor to PdG. There were NOEs between each of the exocyclic ring protons H6a, H7a, and H8a and the T6 CH3 and H6 protons. There was a weak NOE from X7 H6a to T6 H3′. Finally, there was a weak NOE between T8 CH3 and X7 H6b. Chemical Shift Comparisons. Chemical shift comparisons between the -TXT- duplex and the corresponding unmodified duplex suggested that PdG introduced a localized structural perturbation into the DNA (Figure 7). Differences were observed at the adducted base pair X7‚C20 and adjacent base pairs T6‚A21 and T8‚A19. The X7 H2 resonance shifted upfield 0.94 ppm compared to the G7 H8 resonance in the unmodified oligodeoxynucleotide. The T6 H2′ resonance shifted 0.39 ppm upfield and the T8 CH3 and H6 resonances shifted downfield 0.24 and 0.39 ppm, respectively. The T6 H2′ resonance shifted upfield 0.39 ppm, and the C20 H2′ and H2′′ resonances shifted upfield 0.22 and 0.72 ppm, respectively. Structural Refinement. Because the NMR spectra suggested that the sugar at C20 opposite PdG existed in

132

Chem. Res. Toxicol., Vol. 15, No. 2, 2002

Weisenseel et al.

Figure 2. A stacked plot of the data showing the large NOE between X7 H2 f X7 H1′. The presence of this NOE and the missing NOE between T6 H1′ f X7 H2 support the conclusion that base pair X7‚C20 is in the Hoogsteen conformation at pH 5.2.

Figure 3. 1H NOESY spectrum showing resonances from the thymine and guanine imino protons involved in hydrogenbonding interactions. The modified base X7 has no imino proton, so T6 and T8, which flank it on either side show NOEs only to G5 and G18, respectively. The symmetry of the oligodeoxynucleotide is evident, as the spectrum becomes degenerate and the expected sequential connectivities in either strand cannot be traced beyond G4 and G17, respectively.

approximately a 1:1 blend of C2′-endo and C3′-endo conformations, two sets of rMD calculations were performed. In one, C20 was constrained to the C2′-endo conformation, whereas in the other, C20 was constrained to the C3′-endo conformation. For both sets of calculations, a total of 587 distance and 145 dihedral angle restraints were used. There was an average of 28 restraints per nucleotide. The rMD calculations resulted in a set of 20 final structures each for the -TXT- C2′ duplex 〈TXT-C2′-rMD〉, and the -TXT- C3′ duplex 〈TXTC3′-rMD〉. All structure sets were well converged as indicated by the pairwise rmsd comparisons of all 20 structures in each set (〈rMD〉-〈rMD〉) which resulted in rmsd values of 1.06 Å and 1.02 Å for the sets 〈TXT-C2′rMD〉 and 〈TXT-C3′-rMD〉, respectively (Table 1). Overlays of the emergent structures (Figure 8) illustrate the

convergence. Comparison of the final structures by atomic rmsd to the initial A-type and B-type structures (IniA and IniB) indicate that the final structures in each case matched B-type DNA more closely than the A-type DNA. The accuracy of the emergent structures was evaluated by comparison of theoretical NOE intensities calculated by complete relaxation analysis for the average final structures to the experimental intensities to yield sixth root residuals (R1x). The residuals for the structures were below 8.32 × 10-2 and 8.28 × 10-2 for TXT-C2′-rMDavg and TXT-C3′-rMDavg, respectively, indicating that the structures provided an accurate depiction of the data (Table 1). There was little difference in the structures which emerged from rMD calculations constrained to C2′endo and C3′-endo deoxyribose pseudorotation at C20. The most significant difference was in the conformation of the phosphodiester backbone at C20. The C3′-endo structure had a slight kink in the backbone at this position. Overall, the structure of PdG in the -TXT- duplex (Figure 9) revealed the formation of a Hoogsteen base pair between X7 and C20. PdG rotated about the glycosyl bond into the syn conformation. This conformation of PdG placed the propano ring protons in the major groove. The exocyclic ring protons were exposed to solvent. There was a large negative base pair stretch (or compression) at the adduct site due to the rotation of X7 into the syn conformation which left a gap between PdG and the complimentary cytosine. This allowed formation of the Hoogsteen base pair. The Hoogsteen pairing also resulted in a negative base pair opening (or closing), a consequence of the bases rotating toward one another. There was a slight difference in the inter-base-pair shift. As compared to the unmodified duplex, at X7 the phosphodiester backbone angles R and β changed. The angle R shifted to -anticlinal (∼250°) and angle β also shifted to -anticlinal (∼250°) to accommodate formation of the Hoogsteen base pair. This resulted in a kink in the backbone at X7.

Discussion PdG provides a structural model which is stable with respect to ring opening for exocyclic 1,N2-propano lesions of dG (30). This class of lesions is of considerable interest due to the fact that these adducts are formed endog-

Structure of PdG in the -TXT- Sequence

Chem. Res. Toxicol., Vol. 15, No. 2, 2002 133

Figure 4. 1H NOESY spectrum showing the assignment of the exocyclic amino proton resonances of nucleotide C20. These resonances are observed at δ ∼8.7 ppm and δ ∼9.9 ppm. The NOEs between the C20 amino protons and C20 H5 is also shown for reference. The downfield chemical shift of the C20 amino protons is characteristic of Hoogsteen pairing at X7‚C20.

Figure 5. 1H NOESY spectrum showing the assignment of the PdG exocyclic ring protons H6a,b, H7a,b, and H8a,b. The H6a,b protons have the same chemical shift at ∼δ 3.35 ppm. Also shown are NOEs between the exocyclic protons and T6 CH3, T6 H3′, and T6 H1′ the 5′-neighbor nucleotide to PdG. An additional NOE is observed between T8 CH3 and X7 H6a,b. These NOEs position the exocyclic ring of PdG in the major groove of the duplex.

enously, e.g., as a consequence of lipid peroxidation (13). They may also accrue from oxidative damage to DNA via the formation of base propenals (9). Exocyclic 1,N2propanodeoxyguanosine adducts prevent Watson-Crick hydrogen-bonding interactions in DNA. Moreover, the exocyclic ring presents a sterically bulky lesion. NMR studies revealed that PdG disrupted duplex DNA (40, 41,

44, 45). In fact, at neutral pH, it has not been possible to obtain a structure for PdG positioned opposite cytosine in duplex DNA. The spectrum of PdG opposite dC in duplex DNA sharpened considerably as pH was lowered (45). Likewise, when PdG was mismatched with dA in duplex DNA an ordered structure was stabilized at pH 5.8 (41, 42). On the other hand, when PdG was mis-

134

Chem. Res. Toxicol., Vol. 15, No. 2, 2002

Weisenseel et al.

rium observed when PdG is placed opposite from dC or dA in duplex DNA arises from the ability of the PdG base to rotate about the glycosyl bond into the syn conformation. In the syn conformation, PdG can form protonated hydrogen-bonding interactions with dC and dA. When placed opposite dC, this allows formation of a protonated Hoogsteen pairing interaction (45).

Figure 6. Comparison of PdG exocyclic ring proton chemical shifts from the -TXT- duplex to PdG-2BD (46, 47) where the ring protons were embedded in the duplex, PdG 11mer (45) where the ring protons were exposed to solvent, and PdG in single strand DNA. The chemical shifts of the -TXT- duplex most closely resemble the chemical shifts of the PdG 11mer and PdG single strand. This indicates that the PdG protons are exposed to solvent and not stacked in the helix.

matched with dG, an ordered structure was formed which was independent of pH (41). The pH-dependent equilib-

The structural disruption introduced into DNA by PdG suggests that it should be an efficient mutagenic lesion in duplex DNA, and this has proven to be the case. Marnett and co-workers examined PdG in two sequence contexts found in the hisD3052 gene of Salmonella typhimurium (31, 35). One of these, the -CXC- sequence, was contained within an iterated (CG)4 repeat sequence. The other of these, the -TXT- sequence, was found at the center of a palindrome. Both sequences were potential hotspots for frameshift mutations in the hisD3052 gene. The interest in these sequences arose because malondialdehyde acts as a frameshift mutagen in the hisD3052 tester strain of Salmonella typhimurium. The processing of PdG in the -CXC- sequence differed as compared to the -TXT- sequence. In site-specific mutagenesis experiments carried out in E. coli, the mutagenic outcome differed depending upon whether PdG was in the -CGC- sequence or in the -TXT- sequence.

Figure 7. Chemical shift differences when the -TXT- duplex was compared to the corresponding unmodified -TGT- duplex. These indicate a localized structural perturbation of the duplex centered at base pair X7‚C20.

Structure of PdG in the -TXT- Sequence

Chem. Res. Toxicol., Vol. 15, No. 2, 2002 135

Table 1. Statistical Analysis of rMD-Generated Structures for C2′-endo and C3′-endo Conformations of C20 no. of experimental restraints intranucleotide distance restraintst internucleotide distance restraintsa total distance restraints dihedral restraints total experimental restraints structural statistics x)b,c

C2′ endo

C3′ endo

380 207 587 145 732

380 207 587 145 732

C2′ endo

C3′ endo

17.8 10.8 8.28 8.38 8.32

17.8 10.8 8.29 8.41 8.28

10-2)

NMR R-factor (R1 (× IniA IniB rMDAavg rMDBavg rMDavg RMS deviations from the ideal geometry bond length (Å × 10-3) bond angle (deg) improper angle (deg) IniA-IniB 〈rMDA〉-IniA 〈rMDB〉-IniB 〈rMD〉-IniA 〈rMD〉-IniB 〈rMDA〉-〈rMDA〉 〈rMDB〉-〈rMDB〉 〈rMDA〉-〈rMDB〉 〈rMDA〉-rMDavg 〈rMDB〉-rMDavg 〈rMD〉-〈rMD〉 RMSD of NOE violations (× 10-2) avg. no. of NOE violations (>0.1 Å)

8.13 ( 0.03 8.21 ( 0.06 1.560 ( 0.007 1.560 ( 0.006 0.265 ( 0.004 0.259 ( 0.001 6.16 6.02 ( 0.22 1.68 ( 0.12 5.95 ( 0.22 1.69 ( 0.12 0.86 ( 0.28 0.66 ( 0.20 1.32 ( 0.25 0.92 ( 0.15 0.73 ( 0.14 1.06 ( 0.38 2.55 ( 0.03

6.16 6.04 ( 0.20 1.51 ( 0.10 6.00 ( 0.21 1.54 ( 0.11 0.75 ( 0.21 0.62 ( 0.22 1.32 ( 0.24 0.83 ( 0.16 0.79 ( 0.15 1.02 ( 0.39 2.26 ( 0.04

11

14

a Does not include empirical base pairing restraints. b rMDA avg, rMDBavg, and rMDavg are the potential energy minimized average structures for the set of structures resulting from molecular dynamics calculations staring with the A-DNA (rMDAavg), B-DNA (rMDBavg), and both A- and B-DNA (rMDavg). The mixing time used to calculate R1x was 250 ms. c R1x) ∑ |(ao)i1/6 - (ac)i1/6|/∑ |(ao)i1/6|, where (ao) and (ac) are the intensities of observed (nonzero) and calculated NOE cross-peaks, respectively. d The brackets (〈 〉) represent the group of structures.

In the -TXT- sequence, PdG induced both PdG f A and PdG f T mutations (31), whereas in the CGC sequence, PdG did not induce point mutations (35). The replication bypass of PdG lesions in these two sequence contexts in vitro also differed. With the Klenow fragment of DNA polymerase I, PdG produced only 1- and 2-base deletions when placed in the -CXC- sequence. In the -TXTsequence, the predominant product produced by the Klenow fragment was a full-length extension product corresponding to mis-incorporation of dA opposite PdG (39). DNA Sequence Effect. The present studies extend upon the mutagenesis and biochemical studies discussed above and reveal that, at acidic pH, the structural perturbation introduced by PdG into the -TXT- sequence also differs from that introduced into the -CGC- sequence. In both, PdG forms a protonated Hoogsteen base pair with the complementary cytosine at acidic pH. However, in the -TXT- sequence, PdG perturbs only the single base pair at the adduct site. It does not perturb the 3′-neighbor base pair, which remains in the Watson-Crick motif. This contrasted with the structure of PdG in the -CGCsequence was examined at pH 5.8. In that instance PdG also perturbed the 3′-neighbor base pair, which existed in equilibrium between the Watson-Crick and Hoogsteen conformations (45).

Structure of the Protonated PdG‚dC Hoogsteen Pair in the -TXT- Duplex. A distinguishing feature of the PdG lesion in the -TXT- sequence at pH 5.2 is the Hoogsteen pair X7‚C20 which forms at the site of the lesion. The rotation of PdG about the glycosyl bond orients the exocyclic ring protons into the major groove (Figure 9). The syn conformation about the glycosyl bond results in a shorter interproton distance between the H1′ and H2 protons of PdG, evidenced by the large cross-peak between X7 H1′ and H2 (Figure 2). The NOEs between the exocyclic ring protons of PdG and major groove protons of adjacent bases were consistent with the exocyclic ring facing the major groove (Figure 10). The NOEs between X7 H6a, H7a, and H8a to T6 CH3 and H6 would not have been expected had PdG been in the anti conformation. Likewise, an NOE between X7 H6b and T8 CH3 is explained by the positioning of the exocyclic ring of PdG in the major groove. This placed the PdG amino proton X7 H5 near the backbone and exposed to solvent exchange, which was consistent with the failure to observe this proton. Chemical shift evidence also concurred with the orientation of the PdG exocyclic ring protons in the major groove; these shifts were similar to previously observed chemical shifts of PdG in a Hoogsteen pair in the iterated (CG)3 repeat sequence (45) (Figure 6). The downfield shift of the cytosine amino protons was characteristic of protonated cytosine involved in a Hoogsteen base pair (45). An upfield shift of X7 H2 proton of 0.49 ppm was observed by Singh et al. (45), where the PdG adduct Hoogsteen base paired with a N3 protonated cytosine. These chemical shift changes were interpreted to be the result of base stacking and electrostatic changes caused by the rotation of the PdG adduct into the syn conformation and the formation of the X7‚ C20 Hoogsteen base pair. Replication Bypass of PdG in the -TXT- and -CGC- Sequences. (a) Polymerase-Specific Differences. Replication studies carried out in vitro using the Klenow fragment of DNA polymerase I and DNA polymerase β provided insight into the processing of the PdG lesion by DNA polymerases. They showed that the processing of PdG by the two enzymes differed considerably. The Klenow fragment inserted both dA and dG opposite PdG in either the -TXT- or -CXC- sequences. With pol β, the identity of the nucleotide inserted opposite PdG by pol β was dependent upon the identity of the 5′-neighbor nucleotide to PdG. When T was the 5′-neighbor nucleotide as in the -TXT- sequence, pol β preferentially incorporated dA when replicating past PdG (38). (b) Sequence Effects. The consequences of PdG bypass by either the Klenow fragment or pol β were dependent upon DNA sequence. With Klenow, bypass of PdG and formation of full-length extension products was observed only in the -TXT- sequence and not the -CXC- sequence. The major factor enabling full-length replication by the Klenow fragment, in which dA was inserted opposite PdG in the -TXT- sequence, was the preferential extension of template primers containing PdG‚dA termini (39). The observation that dA was incorporated opposite PdG in either the -TXT- or the -CXC- sequence with approximately equal efficiency, but full-length extension was observed only in the -TXT- sequence, suggested that Klenow processed the PdG‚A mismatch differently in the two sequences (39). Deletion products were observed in both the -TXT- and -CXC- sequences. The data suggested that these arose from strand slippage that occurred after

136

Chem. Res. Toxicol., Vol. 15, No. 2, 2002

Weisenseel et al.

Figure 8. Superpositions of structures emergent from the rMD calculations. (A) A set of structures emergent from calculations in which C20 was restrained to the C2′-endo conformation. (B) A set of structures in which C20 was restrained to the C3′-endo conformation.

Figure 10. Detailed view of the -TXT- duplex. The NOEs observed between X7 and T6 and T8 are indicated as dashed lines. These NOEs are consistent with the major groove location of the PdG exocyclic ring.

Figure 9. A view of the -TXT- duplex showing the Hoogsteen pairing at X7‚C20. In this structure, the exocyclic ring of PdG is located in the major groove of the duplex. The Hoogsteen pair is accommodated in the DNA duplex with relatively minor overall structural perturbation.

nucleotide incorporation opposite PdG. However, with pol β, the data suggested strand slippage preceded nucleotide insertion. This suggested that pol β used the dT 5′ to PdG as a template, whereas Klenow used PdG as a template. Structure-Function Implications. The present results showing differences in the low pH structures of the PdG‚C Hoogsteen base pairs in the -CXC- and -TXT-

sequences can be correlated with the observed differences in mutagenesis and replication bypass in these sequences. The presence of sequence specific structural differences in duplex DNA implies that the structures of mismatched or strand slippage structures important in replication bypass of PdG may also differ in the two sequences. For example, an understanding of the structure of the PdG‚A mismatch in the -TXT- sequence as opposed to the -CXC- sequence may be critical to understanding the differential processing of PdG in these two sequences by the Klenow fragment. The structure and stability of the PdG‚A mismatch was examined in the -GXG- sequence at both acidic and basic pH. Structural studies showed that in the -GXG- sequence the mismatch could exist either in the PdG(anti)‚A or PdG(syn)‚A structures, dependent upon pH (40) and documented

Structure of PdG in the -TXT- Sequence

stable PdG (syn)‚A(anti) base pairing (41). Thermodynamic measurements showed the PdG(anti)‚A or PdG(syn)‚A structures exhibited only a 0.4 kcal/mol difference in van’t Hoff free energies at 25 °C (48). The present structural results also have potential implications with regard to frameshift mutations induced by PdG. In the -TXT- sequence, the Klenow fragment induced 1-base deletions by misinsertion of dA followed by slippage of the newly inserted adenine to form the dA‚ dT pair with the 5′-neighbor dT and subsequent extension (39). In contrast, when PdG was in a -CXC- sequence context, 1- and 2-base deletion products were the only products observed during lesion bypasssno point mutations were observed (39). A similar result was reported by Shibutani and Grollman (32). A possible explanation for the sequence dependent differences is that the propensity for strand slippage vs extension from a mismatched primer-template differs in the two sequences. In this regard, the observation that two base pairs corresponding to a single CG repeat unit are perturbed by PdG at pH 5.8 in the CGC sequence (45), whereas in the -TXT- sequence only a single base pair is perturbed, is intriguing. Structural differences for the PdG adduct in the -TXTvs the -CXC- sequences may also imply sequence-specific differences in lesion repair capability. PdG is a substrate for the nucleotide excision repair system in E. coli. It was also excised from a modified 156-mer by a cell free extract from Chinese hamster ovary, suggesting that it is also a substrate for mammalian NER (36). The exact mechanism by which excision repair proteins recognize PdG or other lesions is not well understood, but it may be related to the extent of distortion PdG introduces into duplex DNA.

Summary Structural studies of the PdG lesion in the -TXTsequence context reveal differences as compared to the PdG lesion in the -CXC- sequence context. In both sequences, the exocyclic propano lesion induces structural disorder at neutral pH. At lower pH, PdG forms a protonated Hoogsteen pair with dC in the complementary strand, in both sequences. However, in the -TXT- sequence, the protonated Hoogsteen pair disrupts only the modified base pair, whereas in the -CXC- sequence, it disrupts both the modified base pair and the 3′-neighbor base pair. The structural differences between the two sequences parallel differences in mutagenic outcome and lesion bypass in vitro.

Acknowledgment. This work was supported by NIH Grants CA-55678 (M.P.S.), CA-47479 (L.J.M.), and NIH instrumentation Grant RR-05805 (NMR spectrometer). The Vanderbilt Center Grant in Molecular Toxicology, ES-00267, provided support for laboratory core facilities, including NMR spectroscopy. The University of Wisconsin, NSF Grants DMB-8415048 and BIR-9214394 funded the National Magnetic Resonance Facility at Madison, NIH Grants RR-02301, RR-02781, RR08438, and the USDA. J.P.W. received support from a NIH training grant in molecular biophysics (GM-08320). Supporting Information Available: Tables of chemical shift assignments for the -TGT- and -TXT- duplexes and a table of the analysis of structures of the -TGT- duplex. Figures of the atomic charges on PdG, 1H NOESY spectra of the -TGT- duplex,

Chem. Res. Toxicol., Vol. 15, No. 2, 2002 137 structural superposition of the -TGT- duplex, and stereoviews of -TXT- duplex structures. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Marnett, L. J. (1999) Chemistry and biology of DNA damage by malondialdehyde. IARC Sci. Publ. 150, 17-27. (2) Marnett, L. J. (1999) Lipid peroxidation-DNA damage by malondialdehyde. Mutat. Res. 424, 83-95. (3) Marnett, L. J., and Plastaras, J. P. (2001) Endogenous DNA damage and mutation. Trends Genet. 17, 214-21. (4) Basu, A. K., O’Hara, S. M., Valladier, P., Stone, K., Mols, O., and Marnett, L. J. (1988) Identification of adducts formed by reaction of guanine nucleosides with malondialdehyde and structurally related aldehydes. Chem. Res. Toxicol. 1, 53-59. (5) Marnett, L. J., Basu, A. K., O’Hara, S. M., Weller, P. E., Rahman, A. F. M. M., and Oliver, J. P. (1986) Reaction of malondialdehyde with guanine nucleosides: Formation of adducts containing oxadiazabicyclononene residues in the base-pairing region. J. Am. Chem. Soc. 108, 1348-1350. (6) Seto, H., Okuda, T., Takesue, T., and Ikemura, T. (1983) Reaction of malonaldehyde with nucleic acid. I. Formation of fluorescent pyrimido [1,2-a]purin-10(3H)-one nucleosides. Bull. Chem. Soc. Jpn. 56, 1799-1802. (7) Seto, H., Seto, T., Takesue, T., and Ikemura, T. (1986) Reaction of malonaldehyde with nucleic acid. III. Studies of the fluorescent substances released by enzymatic digestion of nucleic acids modified with malonaldehyde. Chem. Pharm. Bull. 34, 50795085. (8) Reddy, G. R., and Marnett, L. J. (1996) The Mechanism of Reaction of β-aryloxyacroleins with nucleosides. Chem. Res. Toxicol. 9, 12-15. (9) Dedon, P. C., Plastaras, J. P., Rouzer, C. A., and Marnett, L. J. (1998) Indirect mutagenesis by oxidative DNA damage: Formation of the pyrimidopurinone adduct of deoxyguanosine by base propenal. Proc. Natl. Acad. Sci. U.S.A. 95, 11113-11116. (10) Plastaras, J. P., Riggins, J. N., Otteneder, M., and Marnett, L. J. (2000) Reactivity and mutagenicity of endogenous DNA oxopropenylating agents: Base propenals, malondialdehyde, and N(epsilon)-oxopropenyllysine. Chem. Res. Toxicol. 13, 1235-1342. (11) Wang, M. Y., and Liehr, J. G. (1995) Lipid hydroperoxide-induced endogenous DNA adducts in hamsters: Possible mechanism of lipid hydroperoxide-mediated carcinogenesis. Arch. Biochem. Biophys. 316, 38-46. (12) Chaudhary, A. K., Nokubo, M., Reddy, G. R., Yeola, S. N., Morrow, J. D., Blair, I. A., and Marnett, L. J. (1994) Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver. Science 265, 1580-1582. (13) Wang, M., Dhingra, K., Hittleman, W. N., Liehr, J. G., de Andrade, M., and Li, D. (1996) Lipid peroxidation-induced putative malondialdehyde-DNA adducts in human breast tissues. Cancer Epidemiol. 5, 705-710. (14) Nath, R. G., and Chung, F. L. (1994) Detection of exocyclic 1,N2propanodeoxyguanosine adducts as common DNA lesions in rodents and humans. Proc. Natl. Acad. Sci. U.S.A. 91, 7491-7495. (15) Nath, R. G., and Chung, F. L. (1993) Detection of 1,N2-propanodeoxyguanosine adducts in rodent and human liver DNA by 32Ppostlabeling. Proc. Am. Assoc. Cancer Res. 34, 137. (16) O’Nair, J., Barbin, A., Guichard, Y., and Bartsch, H. (1995) 1,N6ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine in liver DNA from humans and untreated rodents detected by immunoaffinity/ 32P-postlabeling. Carcinogenesis 16, 613-617. (17) Chaudhary, A. K., Nokubo, M., Marnett, L. J., and Blair, I. A. (1994) Analysis of the malondialdehyde-2′-deoxyguanosine adduct in rat liver DNA by gas chromatography/electron capture negative chemical ionization mass spectrometry. Biol. Mass Spectrom. 23, 457-464. (18) Rouzer, C. A., Chaudhary, A. K., Nokubo, M., Ferguson, D. M., Reddy, G. R., Blair, I. A., and Marnett, L. J. (1997) Analysis of the malondialdehyde-2′-deoxyguanosine adduct pyrimidopurinone in human leukocyte DNA by gas chromatography/electron capturenegetiave chemical ionization/mass spectrometry. Chem. Res. Toxicol. 10, 181-188. (19) Vaca, C. E., Fang, J. L., Mutanen, M., and Valsta, L. (1995) 32Ppostlabeling determination of DNA adducts of malonaldehyde in humans: Total white blood cells and breast tissue. Carcinogenesis 16, 1847-1851. (20) Fang, J. L., Vaca, C. E., Valsta, L. M., and Mutanen, M. (1996) Determination of DNA adducts of malonaldehyde in humans: Effects of dietary fatty acid composition. Carcinogenesis 17, 10351040.

138

Chem. Res. Toxicol., Vol. 15, No. 2, 2002

(21) Sevilla, C. L., Mahle, N. H., Eliezer, N., Uzieblo, A., O’Hara, S. M., Nokubo, M., Miller, R., Rouzer, C. A., and Marnett, L. J. (1997) Development of monoclonal antibodies to the malondialdehydedeoxyguanosine adduct, pyrimidopurinone. Chem. Res. Toxicol. 2, 172-180. (22) Chaudhary, A. K., Reddy, R. G., Blair, I. A., and Marnett, L. J. (1996) Characterization of an N6-oxopropenyl-2′-deoxyadenosine adduct in malondialdehyde-modified DNA using liquid chromatography/electrospray ionization tandem mass spectrometry. Carcinogenesis 17, 1167-1170. (23) Mao, H., Schnetz-Boutaud, N. C., Weisenseel, J. P., Marnett, L. J., and Stone, M. P. (1999) Duplex DNA catalyzes the chemical rearrangement of a malondialdehyde deoxyguanosine adduct. Proc. Natl. Acad. Sci. U.S.A. 96, 6615-6620. (24) de los Santos, C., Zaliznyak, T., and Johnson, F. (2001) NMR characterization of a DNA duplex containing the major acroleinderived deoxyguanosine adduct γ-OH-1,-N2-propano-2′-deoxyguanosine. J. Biol. Chem. 276, 9077-9082. (25) O’Hara, S. M., and Marnett, L. J. (1991) DNA sequence analysis of spontaneous and β-methoxy-acrolein-induced mutations in Salmonella typhimurium hisD3052. Mutat. Res. 247, 45-56. (26) Streisinger, G., Okada, Y., Enrich, J., Newton, J., Tsugita, A., Terzaghi, E., and Inouye, M. (1966) Frameshift mutations and the genetic code. Cold Spring Harbor Symp. Quantum Biol. 31, 77-84. (27) Fink, S. P., Reddy, G. R., and Marnett, L. J. (1997) Mutagenicity in Escherichia coli of the major DNA adduct derived from the endogenous mutagen malondialdehyde. Proc. Natl. Acad. Sci. U.S.A. 94, 8652-8657. (28) Schnetz-Boutaud, N. C., Saleh, S., Marnett, L. J., and Stone, M. P. (2001) The Exocyclic 1,N2-Deoxyguanosine Pyrimidopurinone M1G is a Chemically Stable DNA Adduct When Placed Opposite a Two Base Deletion in the (CpG)3 Frameshift Hotspot of the Salmonella typhimurium hisD3052 Gene. Biochemistry 40, 1563815649. (29) Mao, H., Reddy, G. R., Marnett, L. J., and Stone, M. P. (1999) Solution structure of an oligodeoxynucleotide containing the malondialdehyde deoxyguanosine adduct N2-(3-oxo-1-propenyl)dG (ring-opened M1G) positioned in a (CpG)3 frameshift hotspot of the Salmonella typhimurium hisD3052 gene. Biochemistry 38, 13491-13501. (30) Marinelli, E. R., Johnson, F., Iden, C. R., and Yu, P. L. (1990) Synthesis of 1,N2-(1,3-propano)-2′-deoxyguanosine and incorporation into oligodeoxynucleotides: A model for exocyclic acroleinDNA adducts. Chem. Res. Toxicol. 3, 49-58. (31) Burcham, P. C., and Marnett, L. J. (1994) Site-specific mutagenesis by a propanodeoxyguanosine adduct carried on an M13 genome. J. Biol. Chem. 269, 28844-28850. (32) Shibutani, S., and Grollman, A. P. (1993) On the mechanism of frameshift (deletion) mutagenesis in vitro. J. Biol. Chem. 268, 11703-11710. (33) Moriya, M., Zhang, W., Johnson, F., and Grollman, A. P. (1994) Mutagenic potency of exocyclic DNA adducts: Marked differences between Escherichia coli and simian kidney cells. Proc. Natl. Acad. Sci. U.S.A. 91, 11899-11903. (34) Fink, S. P., Reddy, G. R., and Marnett, L. J. (1996) Relative contribution of cytosine deamination and error-prone replication to the induction of propanodeoxyguanosine to deoxyadenosine mutations in Escherichia coli. Chem. Res. Toxicol. 9, 277-283. (35) Johnson, K. A. (1995) Genetic requirements for mutations by malondialdehyde in the bacteriophage M13MB102. Ph.D. Dissertation, Vanderbilt University. (36) Johnson, K. A., Fink, S. P., and Marnett, L. J. (1997) Repair of propanodeoxyguanosine by nucleotide excision repair in vivo and in vitro. J. Biol. Chem. 17, 11434-11438. (37) Fink, S. P., and Marnett, L. J. (2001) The relative contribution of adduct blockage and DNA repair on template utilization during replication of 1,N2-propanodeoxyguanosine and pyrimido[1,2]purin-10(3H)-one-adducted M13MB102 genomes. Mutat. Res. 485, 209-218. (38) Hashim, M. F., Schnetz-Boutaud, N., and Marnett, L. J. (1997) Replication of template-primers containing propanodeoxyguanosine by DNA polymerase β. Induction of base pair substitution and frameshift mutations by template slippage and deoxynucleoside triphosphate stabilization. J. Biol. Chem. 272, 20205-20212. (39) Hashim, M. F., and Marnett, L. J. (1996) Sequence-dependent induction of base pair substitutions and frameshifts by propanodeoxyguanosine during in vitro DNA replication. J. Biol. Chem. 271, 9160-9165. (40) Kouchakdjian, M., Eisenberg, M., Live, D., Marinelli, E., Grollman, A. P., and Patel, D. J. (1990) NMR studies of an exocyclic 1, N2-propanodeoxyguanosine adduct (X) located opposite deoxy-

Weisenseel et al.

(41)

(42)

(43)

(44)

(45)

(46)

(47)

(48)

(49) (50)

(51)

(52)

(53) (54)

(55)

(56)

(57)

(58)

(59) (60)

adenosine (A) in DNA duplexes at basic pH: Simultaneous partial intercalation of X and A between stacked bases. Biochemistry 29, 4456-4465. Kouchakdjian, M., Marinelli, E., Gao, X., Johnson, F., Grollman, A., and Patel, D. (1989) NMR studies of exocyclic 1,N2-propanodeoxyguanosine adducts (X) opposite purines in DNA duplexes: Protonated X(syn):A(anti) pairing (acidic pH) and X(syn):G(anti) pairing (neutral pH) at the lesion site. Biochemistry 28, 56475657. Huang, P., and Eisenberg, M. (1992) The three-dimensional structure in solution (pH 5.8) of a DNA 9-mer duplex containing 1,N2-propanodeoxyguanosine opposite deoxyadenosine. Restrained molecular dynamics and NOE-based refinement calculations. Biochemistry 31, 6518-6532. Huang, P., Patel, D. J., and Eisenberg, M. (1993) Solution structure of the exocyclic 1,N2-propanodeoxyguanosine adduct opposite deoxyadenosine in a DNA nonamer duplex at pH 8.9. Model of pH-dependent conformational transition. Biochemistry 32, 3852-3866. Kouchakdjian, M., Eisenberg, M., Johnson, F., Grollman, A. P., and Patel, D. J. (1991) Structural features of an exocyclic adduct positioned opposite an abasic site in a DNA duplex. Biochemistry 30, 3262-3270. Singh, U. S., Moe, J. G., Reddy, G. R., Weisenseel, J. P., Marnett, L. J., and Stone, M. P. (1993) 1H NMR of an oligodeoxynucleotide containing a propanodeoxyguanosine adduct positioned in a (CG)3 frameshift hotspot of Salmonella typhimurium hisd3052: Hoogsteen base-pairing at pH 5.8. Chem. Res. Toxicol. 6, 825-836. Moe, J. G., Reddy, G. R., Marnett, L. J., and Stone, M. P. (1994) 1H NMR characterization of a duplex oligodeoxynucleotide containing propanodeoxyguanosine opposite a two-base deletion in the (CpG)3 frameshift hotspot of Salmonella typhimurium hisD3052. Chem. Res. Toxicol. 7, 319-328. Weisenseel, J. P., Moe, J. G., Reddy, G. R., Marnett, L. J., and Stone, M. P. (1995) Structure of a duplex oligodeoxynucleotide containing propanodeoxyguanosine opposite a two-base deletion in the (CpG)3 frameshift hotspot of Salmonella typhimurium hisD3052 determined by 1H NMR and restrained molecular dynamics. Biochemistry 34, 50-64. Plum, G. E., Grollman, A. P., Johnson, F., and Breslauer, K. J. (1992) Influence of an exocyclic guanine adduct on the thermal stability, conformation, and melting thermodynamics of a DNA duplex. Biochemistry 31, 12096-12102. Borer, P. N. (1975) Handbook of biochemistry and molecular biology. (Fasman, G. D., Ed.) 1st ed., Cleveland, CRC Press. Weisenseel, J. P. (2000) Structural and thermodynamic studies of the hairpins and duplexes formed by a DNA palindrome containing a 1,N2-propanodeoxyguanosine adduct. Ph.D. Dissertation, Vanderbilt University. Piotto, M., Saudek, V., and Sklenar, V. (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Mol. Biol. 6, 661-665. Wang, H., Zuiderweg, E. R. P., and Glick, G. D. (1995) Solution structure of a disulfide cross-linked DNA hairpin. J. Am. Chem. Soc. 117, 2981-2991. Geen, H., and Freeman, R. (1991) Band-selective radiofrequency pulses. J. Magn. Reson. 93, 93-141. Borgias, B. A., and James, T. L. (1990) MARDIGRAS- -a procedure for matrix analysis of relaxation for discerning geometry of an aqueous structure. J. Magn. Reson. 87, 475-487. Liu, H., Tonelli, M., and James, T. L. (1996) Correcting NOESY cross-peak intensities for partial relaxation effects enabling accurate distance information. J Magn. Reson. B 111, 85-89. Liu, H., Spielmann, H. P., Ulyanov, N. B., Wemmer, D. E., and James, T. L. (1995) Interproton distance bounds from 2D NOE intensities: Effect of experimental noise and peak integration errors. J Biomol. NMR 6, 390-402. Rinkel, L. J., and Altona, C. (1987) Conformational analysis of the deoxyribofuranose ring in DNA by means of sums of protonproton coupling constants: A graphical method. J. Biomol. Struct. Dyn. 4, 621-649. Lankhorst, P. P., Haasnoot, C. A. G., Erkelens, C., and Altona, C. (1984) Carbon-13 NMR in conformational analysis of nucleic acid fragments 2. A reparametrization of the Karplus equation for vicinal NMR coupling constants in CCOP and HCOP fragments. J. Biomol. Struct. Dyn. 1, 1387-1405. Gorenstein, D. G. (1992) 31P NMR of DNA. Methods Enzymol. 211, 254-286. Altona, C. (1982) Conformational analysis of nucleic acids. Determination of backbone geometry of single-helical RNA and DNA in aqueous solution. Recl. Trav. Chim. Pays-Bas 101, 413433.

Structure of PdG in the -TXT- Sequence (61) Arnott, S., and Hukins, D. W. L. (1972) Optimised parameters for A-DNA and B-DNA. Biochem. Biophys. Res. Commun. 47, 1504-1509. (62) Stewart, J. P. (1983) MOPAC. Quantum Chem. Prog. Bull. 3, 43. (63) Brunger, A. T. (1992) X-Plor. Version 3.1. A system for X-ray Crystallography and NMR, New Haven, Yale University Press. (64) Nilsson, L., and Karplus, M. (1986) Empirical energy functions for energy minimization and dynamics of nucleic acids. J. Comput. Chem. 7, 591-616. (65) Clore, G. M., Gronenborn, A. M., Carlson, G., and Meyer, E. F. (1986) Stereochemistry of binding of the tetrapeptide acetyl-proala-pro-tyr-NH2 to porcine pancreatic elastase. Combined use of two-dimensional transferred nuclear Overhauser enhancement measurements, restrained molecular dynamics, X-ray crystallography and molecular modeling. J. Mol. Biol. 190, 259-267. (66) Ryckaert, J.-P., Ciccotti, G., and Berendsen, H. J. C. (1977) Numerical integration of the Cartesian equations of motion of a system with constraints: Molecular dynamics of n-alkanes. J. Comput. Phys. 23, 327-341. (67) Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A., and Haak, J. R. (1984) Molecular dynamics with coupling to an external bath. J. Phys. Chem. 81, 3684-3690.

Chem. Res. Toxicol., Vol. 15, No. 2, 2002 139 (68) Keepers, J. W., and James, T. L. (1984) A theoretical study of distance determination from NMR. Two-dimensional nuclear Overhauser effect spectra. J. Magn. Reson. 57, 404-426. (69) Liu, Y., Zhao, D., Altman, R., and Jardetzky, O. (1992) A systematic comparison of three structure determination methods from NMR data: Dependence upon quality and quantity of data. J. Biomol. NMR 2, 373-388. (70) Thomas, P. D., Basus, V. J., and James, T. L. (1991) Protein solution structure determination using distances from twodimensional nuclear Overhauser effect experiments: Effect of approximations on the accuracy of derived structures. Proc. Natl. Acad. Sci. U.S.A. 88, 1237-1241. (71) Ravishankar, G., Swaminathan, S., Beveridge, D. L., Lavery, R., and Sklenar, H. (1989) Conformational and helicoidal analysis of 30 ps of molecular dynamics on the d(CGCGAATTCGCG) double helix: “Curves”, dials, and windows. J. Biomol. Struct. Dyn. 6, 669-699. (72) Reid, B. R. (1987) Sequence-specific assignments and their use in NMR studies of DNA structure. Q. Rev. Biophys. 20, 2-28.

TX0101090