Bulge Migration of the Malondialdehdye OPdG DNA Adduct When Placed Opposite a Two-Base Deletion in the (CpG)₃ Frameshift Hotspot of the Salmonella typhimurium hisD3052 Gene

Abstract

The OPdG adduct N²-(3-oxo-1-propenyl)dG, formed in DNA exposed to malondialdehyde, was introduced into 5′-d(ATCGCXCGGCATG)-3′•5′-d(CATGCCGCGAT)-3′ at pH 7 (X = OPdG). The OPdG adduct is the base-catalyzed rearrangement product of the M₁dG adduct, 3-(β-d-ribofuranosyl)pyrimido[1,2-a]purin-10(3H)-one. This duplex, named the OPdG-2BD oligodeoxynucleotide, was derived from a frameshift hotspot of the Salmonella typhimuium hisD3052 gene and contained a two-base deletion in the complementary strand. NMR spectroscopy revealed that the OPdG-2BD oligodeoxynucleotide underwent rapid bulge migration. This hindered its conversion to the M₁dG-2BD duplex, in which the bulge was localized and consisted of the M₁dG adduct and the 3′-neighbor dC [Schnetz-Boutaud, N. C., Saleh, S., Marnett, L. J., and Stone, M. P. (2001) Biochemistry 40, 15638−15649]. The spectroscopic data suggested that bulge migration transiently positioned OPdG opposite dC in the complementary strand, hindering formation of the M₁dG-2BD duplex, or alternatively, reverting rapidly formed intermediates in the OPdG to M₁dG reaction pathway when dC was placed opposite from OPdG. The approach of initially formed M₁dG-2BD or OPdG-2BD duplexes to an equilibrium mixture of the M₁dG-2BD and OPdG-2BD duplexes was monitored as a function of time, using NMR spectroscopy. Both samples attained equilibrium in ∼140 days at pH 7 and 25 °C.

Introduction

The exocyclic guanine adduct M₁dG ¹ [3-(2′-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one] arises in DNA from multiple sources. One source is exposure to malondialdehyde (MDA), a toxic and mutagenic metabolite produced by lipid peroxidation and prostaglandin biosynthesis (for a review, see refs ¹ and ²). In aqueous solution, MDA exists as β-hydroxyacrolein. It reacts with DNA as a bis-electrophile to form M₁dG (3–7). Alternatively, the M₁dG adduct arises as a consequence of oxidative damage to DNA, resulting in the formation of base propenals that subsequently transfer their oxopropenyl group to dG (8) (Scheme 1). The base propenals are significantly more potent than MDA in forming M₁dG (9).

Scheme 1.

(A) Formation of M₁dG from MDA or from Base Propenals and (B) Depiction of M₁dG Being Stable in Single-Stranded DNA ^a

The M₁dG adduct has been identified in DNA from rodent (10) and human (11, 12) tissue samples, as have other exocyclic purine lesions (13–16), suggesting their presence in vivo. The levels of M₁dG adducts in DNA have been quantitated by mass spectrometric (17, 18), postlabeling (19, 20), and immunochemical (21) techniques. This endogenously formed adduct is the most abundant exocyclic adduct present in human DNA (18–20, 22). It has also been detected at low levels in human urine (23). The low urinary levels of M₁dG likely reflect metabolic conversion to the 6-oxo-M₁dG derivative (24). M₁dG is an efficient premutagenic lesion in Escherichia coli (25, 26), mammalian (26), and human (27) cells. Thus, the M₁dG adduct is anticipated to mediate human carcinogenesis.

The M₁dG adduct is stable in nucleotides and single-stranded DNA at neutral pH. Under basic conditions, it converts to the N²-(3-oxo-1-propenyl)dG (OPdG) derivative. In contrast, when M₁dG is placed at neutral pH into duplex DNA opposite dC, a spontaneous conversion to the OPdG derivative is facilitated. Upon denaturation of the duplex, M₁dG is regenerated (Scheme 2). Ring opening does not occur at neutral pH in duplex DNA if thymine is placed opposite from M₁dG. These observations suggested that dC in duplex DNA catalyzes the transformation of M₁dG to its ring-opened OPdG derivative (28). The ring opening of M₁dG as a nucleoside or in oligodeoxynucleotides is a reversible second-order reaction with hydroxide ion (29). The reverse ring closure mechanism involves rapid formation of protonated OPdG and 8-hydroxy-6,7-propenodG intermediates that slowly converts to M₁dG in a general acid-catalyzed reaction (30).

Scheme 2.

2BD Oligodeoxynucleotide Duplex Containing a Two-Nucleotide 5′-GpC-3′ Deletion in the Complementary Strand ^a

A refined structure was obtained for the OPdG adduct in 5′-d(ATCGCXCGGCATG)-3′•5′-d(CATGCCGCGCGAT)-3′ (X =OPdG) (31). This sequence contains the d(CpG)₃ frameshift mutation hotspot located in the Salmonella typhimurium hisD3052 gene. The structure of the OPdG adduct embedded in this sequence was of interest because MDA, a small alkylating agent, induced frameshift mutations (32). In this structure, OPdG maintained stacking interactions with neighboring bases. It was not Watson–Crick hydrogen bonded. The cytosine complementary to OPdG was pushed toward the major groove but maintained partial stacking with its neighboring bases. The modified guanine remained in the anti conformation, while the OPdG 3-oxo-1-propenyl moiety was positioned in the minor groove of the duplex (31).

The frameshift mutations observed in vivo in E. coli and in COS-7 cells and associated with the d(CpG)₃ iterated repeat included −2 bp deletions (26). Thus, it was of interest to examine oligodeoxynucleotides containinga2bp (CpG) deletion opposite either M₁dG or OPdG. Additionally, replication bypass studies conducted in vitro suggested that DNA polymerase β induced two-base deletions when replicating past M₁dG in this iterated sequence derived from the S. typhimuium hisD3052 gene. ² Previous NMR structural studies examined the M₁dG adduct in the 5′-d(A⁻²T⁻¹C¹G²C³X⁴C⁵G⁶G⁷C⁸A⁹T¹⁰G¹¹)-3′•5′-d(C¹²A¹³T¹⁴G¹⁵C¹⁶C¹⁷G¹⁸C¹⁹G²⁰A²¹T²²)-3′ sequence containing a 2 bp deletion in the complementary strand (Scheme 2), named the M₁dG-2BD oligodeoxynucleotide (33). In that freshly prepared sample, the two-base bulge in the M₁dG-2BD oligodeoxynucleotide was reported to be localized and consisted of M₁dG and the 3′-neighbor dC. The structure of the M₁dG-2BD duplex suggested that the bulged sequence lacked a cytosine properly positioned to facilitate opening of M₁dG and supported the conclusion that proper positioning of dC complementary to M₁dG is necessary for promotion of ring opening of the exocyclic adduct in duplex DNA (33). The structure of the M₁dG-2BD duplex was similar to that of the structural analogue 1,N²-propanodeoxyguanosine (PdG) in the corresponding PdG-2BD duplex (34). The fixed position of the bulged bases in both instances suggested (33) that these exocyclic adducts did not facilitate bulge migration, a phenomenon first reported by Woodson and Crothers (35, 36).

The NMR studies presented here examine the OPdG-2BD oligodeoxynucleotide, in which the OPdG adduct is placed opposite the two-nucleotide deletion (Scheme 1). In contrast to the M₁dG-2BD oligodeoxynucleotide (33), the OPdG-2BD oligodeoxynucleotide undergoes rapid bulge migration (35, 36). This hinders its conversion to the M₁dG-2BD duplex (33). The data suggest that bulge migration transiently positions OPdG opposite dC in the complementary strand or, alternatively, reverts rapidly formed intermediates in the OPdG to M₁dG reaction pathway (30) when dC is placed opposite from OPdG. In contrast, the localized bulge in the M₁dG-2BD duplex consisting of the M₁dG adduct and the 3′-neighbor dC (33) hinders the conversion of M₁dG to OPdG that is catalyzed by the positioning of cytosine opposite from M₁dG (28, 29). The addition of either M₁dG-2BD- or OPdG-2BD-containing samples to an equilibrium mixture was monitored as a function of time, using NMR spectroscopy. Both samples attained equilibrium, slightly favoring the M₁dG-2BD duplex, in ∼140 days at pH 7 and 25 °C.

Materials and Methods

Oligodeoxynucleotide Synthesis

The unmodified oligodeoxynucleotide was synthesized by the Midland Certified Reagent Co. (Midland, TX) and purified by anion-exchange chromatography. M₁dG was synthesized, purified, and incorporated into oligodeoxynucleotides using either an older (37, 38) or a more recently described and improved methodology (39). The extinction coefficients of the oligodeoxynucleotides were calculated on the basis of nearest-neighbor analysis and were 1.04 × 10⁵ M⁻¹ cm⁻¹ for the M₁dG-modified strand 5′-d(ATCGCXCGGCATG)-3′ and 1.01 × 10⁵ M⁻¹ cm⁻¹ for the complementary strand 5′-d(CATGCCGCGAT)-3′ (40). To prepare the OPdG-2BD duplex, the 13-mer containing the M₁dG adduct was combined with the 11-mer complementary strand at a 1:1 molar ratio in 0.1 M NaCl, 10 mM NaH₂PO₄, and 50 μM Na₂EDTA at pH 11.8. After 3 h at room temperature, the buffer was rapidly adjusted to pH 7.0. The M₁dG-2BD duplex was prepared as previously described (33). For spectroscopic examination of the nonexchangeable protons, the OPdG-2BD and M₁dG-2BD samples were then lyophilized and exchanged three times with D₂O and then suspended in 99.996% D₂O. Samples used for spectroscopic examination of exchangeable protons were suspended in a 9:1 H₂O/D₂O mixture. The concentrations of both the OPdG-2BD and M₁dG-2BD duplex samples were 0.52 mM. The concentration of the unmodified duplex sample was 1.1 mM.

NMR Spectroscopy

¹H NMR spectra were recorded at 500.13 and 800.13 MHz. Chemical shifts were referenced to the water resonance. Phase sensitive NOESY spectra used for resonance assignments were recorded at 25 ± 0.5 °C using TPPI phase cycling with a mixing time of 250 ms. For examination of exchangeable protons, phase sensitive NOESY experiments were carried out using a field gradient Watergate pulse sequence for water suppression (41). The spectra were recorded at 10 ± 0.5 °C with a mixing time of 150 ms. These experiments were generally recorded with 512 real data points in the d₁ dimension and 2048 real data points in the d₂ dimension. A relaxation delay of 1.9 s was used for these experiments. The data were processed using FELIX (Accelrys, Inc., San Diego, CA) on Octane workstations (Silicon Graphics, Inc., Mountain View, CA). The data in the d₁ dimension were zero-filled to give a matrix of 2048 × 2048 real points. A skewed sine-bell square apodization function with a 90° phase shift and a skew factor of 1.0 was used in both dimensions.

Results

¹H Resonance Assignments of Nonexchangeable DNA Protons for the Unmodified 2BD Duplex

The unmodified 2BD duplex contained eight cytosines. These each exhibited sharp COSY cross-peaks arising from H5–H6 scalar couplings, shown in Figure 1A. Although there was spectral overlap arising from the iterated C¹G²C³G⁴C⁵G⁶ repeat sequence, at 800 MHz it was possible to resolve resonances with slightly different chemical shifts. Thus, the C³ H6 resonance was located at 7.35 ppm, slightly downfield of C¹⁷ H6, located at 7.36 ppm. The C³ H5 resonance was located at 5.46 ppm, slightly upfield from the C¹⁷ H5 resonance, located at 5.49 ppm. The C⁵ H6 resonance was located at 7.19 ppm, slightly upfield of the C¹⁹H6 resonance located at 7.22 ppm. The C⁸ H6 resonance was located at 7.28 ppm, slightly upfield of the C¹⁶ H6 resonance located at 7.31 ppm.

Figure 1.

(A) COSY spectrum of the unmodified 2BD duplex. (B) COSY spectrum of the freshly prepared OPdG-2BD duplex. (C) COSY spectrum of the freshly prepared M₁dG-2BD duplex. Cross-peaks: a, C¹² H5 → C¹² H6; b, C¹ H5 → C¹ H6; c, C³ H5 → C³ H6; d, C¹⁷ H5 → C¹⁷ H6; e, C¹⁶ H5 → C¹⁶ H6; f, C⁸ H5 → C⁸ H6; g, C⁵ H5 → C⁵ H6; h, C¹⁹ H5 → C¹⁹ H6; i, M₁dG H7 → H8; j, M₁dG H6 → H7. The experiments were performed at 800 MHz and 25 °C.

An expanded plot from the NOESY spectrum for the unmodified 2BD duplex, showing the sequential aromatic–anomeric proton connectivities, is also shown in Figure 2 A,B. The NOESY data were also characterized by sharp cross-peaks. The sequential NOE assignments were made using standard protocols (42, 43). The A⁻² H1′ → T⁻¹ H6 cross-peak was weak, reflecting fraying at the terminal A⁻²•T²² base pair. The T²² H1′ resonance was located at 6.01 ppm. The T⁻¹ H1′ resonance was located at 5.97 ppm, the C¹ H1′ resonance at 5.92 ppm, and the G² H1′ resonance at 5.94 ppm. In the 13-mer strand, this resulted in overlap of the T⁻² H6 → T⁻¹ H1′ and G² H1′ → C³ H6, T⁻¹ H1′ → C¹ H6 and C¹ H6 → C¹ H1′, and C¹ H1′ → G² H8 and G² H8 → G² H1′ NOESY cross-peaks. The C³ H6 → C³ H1′ cross-peak was partially superimposed upon the stronger C³ H5 → C³ H6 and C¹⁷ H5 → C¹⁷ H6 cross-peaks. The G⁴ H8 resonance was located at 7.87 ppm, slightly downfield of G¹⁸ H8, located at 7.84 ppm. This caused the G⁴ H8 → G⁴ H1′ and G¹⁸ H8 → G¹⁸ H1′ cross-peaks to be superimposed. The G⁴ H8 → C⁵ H6 cross-peak overlapped with the G¹⁸ H8 → C¹⁹ H6 cross-peak, and the C⁵ H6 → C⁵ H1′ cross-peak overlapped with the C¹⁹ H6 → C¹⁹ H1′ cross-peak. The C¹⁹ H1′ resonance was located at 5.58 ppm, slightly downfield of G²⁰ H1′, located at 5.45 ppm. The chemical shifts of the nonexchangeable protons are listed in Table S1 of the Supporting Information.

Figure 2.

(A) Expanded plot showing sequential NOE connectivity between aromatic and anomeric protons for nucleotides A⁻² → G¹¹ of the unmodified 2BD duplex. (B) Expanded plot showing sequential NOE connectivity between aromatic and anomeric protons for nucleotides C¹² → T²² of the unmodified 2BD duplex. (C) Expanded plot showing sequential NOE connectivity between aromatic and anomeric protons for nucleotides A⁻² → G¹¹ of the OPdG-2BD duplex. (D) Expanded plot showing sequential NOE connectivity between aromatic and anomeric protons for nucleotides C¹² → T²² of the OPdG-2BD duplex. The 800 MHz 250 ms mixing time NOESY experiments were conducted at 25 °C.

¹H Resonance Assignments of Exchangeable DNA Protons for the Unmodified 2BD Duplex

The far-downfield region of the NOESY spectrum showing the exchangeable resonances arising from the Watson–Crick base-paired imino protons is shown in Figure 3A. A complete sequential connectivity was traced in the noniterated portion of the oligodeoxynucleotide duplex, from G⁶ N1H → G⁷ N1H → G¹⁵ N1H → T¹⁴ N3H → T¹⁰ N3H using standard protocols (44). The G¹¹ N1H resonance was not observed, presumably due to fraying of the G¹¹•C¹² terminal base pair, accompanied by rapid exchange with solvent. There were no unusual chemical shifts noted among this group of resonances. The imino resonance for the A⁻²•T²² terminal base pair at the other end of the oligodeoxynucletoide duplex was also not observed, presumably due to rapid exchange with solvent. In the reiterated portion of the 2BD duplex, the imino resonance arising from T⁻¹ N3H was also not observed, suggesting its rapid exchange with solvent. In addition, a broad resonance, assigned as one or more of the imino proton resonances arising from nucleotides G²⁰, G¹⁸, G², and G⁴, was observed at ∼12.9 ppm. Consequently, the imino resonances from nucleotides T⁻¹, and nucleotides G²⁰, G¹⁸, G², and G⁴, located in the iterated repeat portion of the duplex, could not be unequivocally identified. The exocyclic N⁴ amino protons of cytosines exhibit longer lifetimes with respect to solvent exchange than the N3 imino protons. Inspection of the amino region of the ¹H NMR spectrum (Figure 3A) revealed the presence of resonances arising from hydrogen-bonded and non-hydrogen-bonded exocyclic amino protons of nucleotides C¹, C¹⁹, C³, and C⁵. This indicated that on the NMR time scale, each of the nucleotides (C¹, C¹⁹, C³, and C⁵) was involved in Watson–Crick hydrogen bonding with a deoxyguanosine in the complementary strand. Thus, the nucleotides in the complementary strand, G¹⁸, C¹⁹, and G²⁰, must be transiently forming Watson–Crick hydrogen bonds with C¹, G², C³, G⁴, and C⁵ in the parent strand, i.e., bulge migration (35) (see Scheme 2). No cross-peaks were observed between the hydrogen-bonded and non-hydrogen-bonded N⁴ exocyclic amino protons of C¹ and deoxyguanosine imino protons. Furthermore, the chemical shift difference between the hydrogen-bonded and non-hydrogen-bonded amino protons of C¹ was smaller than that observed for the other deoxycytosine residues, with the exception of terminal deoxycytosine residue C¹². This observation suggested that on the NMR time scale, nucleotides C¹ and G² were preferentially unpaired as compared to nucleotides G⁴ and C⁵. In effect, the unmodified duplex was undergoing sequential fraying from the 5′-terminus of the 13-mer strand, extending into the duplex and involving the A⁻²•T²², T⁻¹•A²¹, C¹•G²⁰, and G²•C¹⁹ base pairs. The chemical shifts of the exchangeable protons are listed in Table S4 of the Supporting Information.

Figure 3.

(A) Expanded plot showing sequential NOE connectivity of the base-paired imino protons for the unmodified 2BD duplex. (B) Expanded plot showing sequential NOE connectivity for the base-paired imino protons of the freshly prepared OPdG-2BD duplex. Cross-peaks: a, C¹⁶ N⁴H2 → C¹⁶ H5; b, C¹⁶ H6 → C¹⁶ H5; c, C¹⁶ N⁴H1 → C¹⁶ H5; d, C⁸ N⁴H2 → C⁸ H5; e, C⁸ H6 → C⁸ H5; f, C⁸ N⁴H1 → C⁸ H5; g, C⁵ N⁴H2 → C⁵ H5; h, C⁵ H6 → C⁵ H5; i, C⁵ N⁴H1 → C⁵ H5; j, C¹⁹ N⁴H2 → C¹⁹ H5; k, C¹⁹ H6 → C¹⁹ H5; l, C¹⁹ N⁴H1 → C¹⁹ H5; m, C¹⁷ N⁴H2 → C¹⁷ H5; n, C¹⁷ H6 → C¹⁷ H5; o, C¹⁷ N⁴H1 → C¹⁷ H5; p, C³ N⁴H2 → C³ H5; q, C³ H6 → C³ H5; r, C³ N⁴H1 → C³ H5; s, C¹ N⁴H2 → C¹ H5; t, C¹ H6 → C¹ H5; u, C¹ N⁴H1 → C¹ H5; v, C¹² N⁴H2 → C¹² H5; w, C¹² H6 → C¹² H5; x, C¹² N⁴H1 → C¹² H5; y, G¹⁵ N1H → C⁸ N⁴H2; z, G¹⁵ N1H → C⁸ N⁴H1; aa, G⁷ N1H → C¹⁶ N⁴H2; bb, G⁷ N1H → C¹⁶ N⁴H1; cc, G¹⁸ N1H → C⁵ N⁴H2; dd, G² N1H and X⁴ N1H → C¹⁹ N⁴H2; ee, G⁶ N1H and X⁴ N1H → C¹⁷ N⁴H2; ff, G¹⁸ N1H → C⁵ N⁴H1; gg, G² N1H and X⁴ N1H → C¹⁹ N⁴H1; hh, G⁶ N1H and X⁴ N1H → C¹⁷ N⁴H1; ii, G¹⁸ N1H and G²⁰ N1H → C³ N⁴H2; jj, G¹⁸ N1H and G²⁰ N1H → C³ N⁴H1. The 800 MHz, 250 ms mixing time NOESY experiments were performed at 10 °C.

¹H Resonance Assignments of Nonexchangeable DNA Protons for the OPdG-2BD Duplex

As compared to those of the unmodified 2BD duplex, the resonances arising from the iterated C¹G²C³G⁴C⁵G⁶ repeat sequence of the OPdG-2BD duplex exhibited better resolution. The eight cytosines each exhibited sharp COSY cross-peaks arising from H5–H6 scalar couplings, as shown in Figure 1 B.

The sequential NOE assignments shown in Figure 2 C,D were made using standard protocols (42, 43). No disruptions in sequential NOE connectivity were observed in either the modified stand or the complementary strand. In the modified 13-mer strand, the A⁻² H1′ → T⁻¹ H6, T⁻¹ H6 → T⁻¹ H1′, and T⁻¹ H1′ → G² H8 cross-peaks were weak, probably reflecting an increased level of fraying at terminal A⁻²•G²² and T⁻¹•A²¹ base pairs. The C⁵ H1′ → G⁶ H8 cross-peak was also weak. In the complementary strand, the C¹⁶ H6 resonance was observed at 7.23 ppm while the C¹⁷ H6 resonance was observed at 7.30 ppm. The G¹⁵ H1′ resonance was observed at 5.73 ppm. This was slightly upfield of the C¹⁶ H1′ resonance. The G¹⁶ H1′ resonance was observed at 5.73 ppm. This caused both the G¹⁵ H1′ → C¹⁶ H6 and C¹⁶ H6 → C¹⁶ H1′ cross-peaks to be superimposed with the G¹⁸ H1′ → C¹⁹ H6 and C¹⁹ H6 → C¹⁹ H1′ cross-peaks. No unusual ¹H shifts were noted in this region of the spectrum. The C¹⁹ H1′ → G²⁰ H8 and G²⁰ H1′ → A²¹ H8 cross-peaks were weak, probably due to fraying at the terminal base pairs. The chemical shifts of the nonexchangeable protons are listed in Table S2 of the Supporting Information.

¹H Resonance Assignments of Exchangeable DNA Protons for the OPdG-2BD Duplex

The far-downfield region of the NOESY spectrum showing the exchangeable resonances arising from the Watson–Crick base-paired imino protons is shown in Figure 3 B. Significantly, it appeared substantially similar to the corresponding spectrum in Figure 3 A for the nonadducted 2BD duplex. For the non-reiterated portion of the duplex, sequential connectivity was traced from G⁶ N1H → G⁷ N1H → G¹⁵ N1H → T¹⁴ N3H → T¹⁰ N3H using standard protocols (44). There were no unusual chemical shifts noted among this group of resonances. The G¹¹ N1H resonance was not observed, presumably due to fraying of the G¹¹•C¹² terminal base pair, accompanied by rapid exchange with solvent. At the other end of the duplex, the imino resonance for the A⁻²•T²² base pair was also not observed, suggesting rapid exchange with solvent. Additionally, the T⁻¹ N3H resonance was not observed, presumably due to rapid exchange with solvent. A broad resonance, assigned as one or more of the imino proton resonances arising from nucleotides G²⁰, G¹⁸, G², and X⁴, was observed at ∼12.9 ppm. Consequently, the imino resonances from nucleotides T⁻¹, and nucleotides G²⁰, G¹⁸, G², and X⁴, located in the iterated repeat portion of the duplex, could not be unequivocally identified. Inspection of the amino region of the ¹H NMR spectrum (Figure 3B) revealed weaker resonances arising from hydrogen-bonded and non-hydrogen-bonded exo-cyclic amino protons of nucleotides C¹, C¹⁹, C³, and C⁵, as compared to the unmodified 2BD duplex (Figure 3A). This was attributed, in part, to the adducted sample being less concentrated but, more significantly, to slower bulge migration in the OPdG-2BD duplex as compared to the unmodified 2BD duplex, resulting in exchange broadening. Nevertheless, as in the unmodified 2BD duplex, on the NMR time scale, each of the nucleotides (C¹, C¹⁹, C³, and C⁵) exhibited evidence of being involved in Watson–Crick hydrogen bonding with a deoxyguanosine in the complementary strand. Thus, the nucleotides in the complementary strand, G¹⁸, C¹⁹, and G²⁰, must be transiently forming Watson–Crick hydrogen bonds with C¹, G², C³, X⁴, and C⁵, in the parent strand (see Scheme 2). No cross-peaks were observed between the hydrogen-bonded and non-hydrogen-bonded N⁴ exocyclic amino protons of C¹ and deoxyguanosine imino protons. However, the chemical shift difference between the hydrogen-bonded and non-hydrogen-bonded amino protons of C¹ was now greater than that observed for C¹ in the unmodified 2BD duplex (Figure 3A), approaching that observed for the other deoxycytosine residues, with the exception of terminal deoxycytosine residue C¹². This suggested that on the NMR time scale, nucleotides C¹ and G² were paired more frequently than in the unmodified 2BD duplex; i.e., the presence of the OPdG lesion at X⁴ resulted in somewhat more favorable paring of base pairs C¹•G²⁰ and G²•C¹⁹ than was the case in the unmodified 2BD duplex, but the OPdG-2BD duplex maintained a more rapid rate of bulge migration than did the M₁dG-2BD duplex, where the bulge was clearly localized at nucleotides X⁴ and C⁵. The chemical shifts of the exchangeable protons are listed in Table S4 of the Supporting Information.

Assignments of the OPdG Protons and NOEs between OPdG and DNA in the OPdG-2BD Duplex

The OPdG proton resonances were identified from a combination of NOESY and COSY experiments. Figure 4 shows the chemical shifts of OPdG protons and intermolecular NOEs between OPdG adduct protons and DNA. OPdG H8 was furthest downfield. At 8.96 ppm, it showed a strong NOE to OPdG H6 located at 7.95 ppm and a broad NOE to OPdG H7 located at 5.72 ppm. The OPdG H6 proton showed a broad NOE to OPdG H7. Six NOEs were observed between OPdG protons and DNA. All of these involved the adducted nucleotide X⁴ and 3′-neighbor nucleotide C⁵ in the modified strand. Both OPdG H8 and H6 exhibited NOEs to C⁵ H5′ and C⁵ H1′; OPdG H8 also exhibited a NOE to C⁵ H4′ and X⁴ H1′.

Figure 4.

Tile plot showing NOE cross-peaks between OPdG protons and DNA protons in the OPdG-2BD duplex. Cross-peaks: a, OPdG H8 → C⁵ H5′; b, OPdG H8 → C⁵ H4′; c, OPdG H8 → C⁵ H1′; d, OPdG H8 → OpdG H6; e, OPdG H8 → X⁴ H1′; f, OPdG H8 → OPdG H7; g, OPdG H8 → OPdG H8; h, OPdG H7 → C⁵ H5′; i, OPdG H7 → C⁵ H1′; j, OPdG H7 → OPdG H6. The 800 MHz, 250 ms mixing time NOESY experiment was conducted at 25 °C.

¹H Resonance Assignments of the M₁dG-2BD Duplex

The detailed NMR assignments of the M₁dG-2BD duplex were as previously reported (33). In contrast to the OPdG-2BD duplex and to the unmodified 2BD duplex, disruptions in sequential NOE connectivity were observed in both the modified and complementary strands. In the modified strand, the C³ H1′ → X⁴ H2 sequential NOE (X⁴ H2 is the imidazole proton of M₁dG, corresponding to G⁴ H8 in the unmodified nucleotide) was missing. In the complementary strand, the C¹⁷ H1′ → G¹⁸ H8 NOE was not observed. Spectral line broadening was localized adjacent to the M₁dG lesion. Line broadening was observed for the C³ and C⁵ H5 and H6 resonances. These were the 5′- and 3′-neighbors of the M₁dG lesion, respectively. In the complementary strand, line broadening was observed for C¹⁹ H5 and H6 (Figure 1 C). The assignments for the base imino protons of the M₁dG-2BD duplex showed an interruption in the sequential connectivity between imino protons at G¹⁸, the base pair 5′ to the M₁dG lesion. No NOE connectivity was observed between G¹⁸ N1H and G⁶ N1H, which localized the M₁dG adduct between base pairs C⁵•G¹⁸ and G⁶•C¹⁷. The exocyclic M₁dG protons H6, H7, and H8 of M₁G were identified as the characteristic aromatic spin system. M₁dG H8 was observed at 8.4 ppm, M₁dG H7 at 5.8 ppm, and M₁dG H6 at 8.2 ppm. The following ³J coupling constants for the M₁dG protons were measured in a DQF-COSY experiment: ³J_H6,H7 ∼ 4 Hz and ³J_H7,H8 ∼ 7 Hz. Figure 5 A shows chemical shift comparisons of cytosine protons H5 and H6, between the OPdG-2BD duplex and the unmodified 2BD duplex. No chemical shift perturbations were greater than 0.1 ppm except at position C¹⁶ H5 where a downfield shift of 0.24 ppm was observed. Figure 5 B shows chemical shift comparisons of cytosine proton H5–H6 cross-peaks, between the M₁dG-2BD duplex and the unmodified 2BD duplex. No chemical shift perturbations were greater than 0.1 ppm except at position C⁵ in the modified strand and C¹⁹ in the complementary strand. A downfield shift of 0.44 ppm was observed for the C⁵ H5 resonance, a downfield shift of 0.21 ppm for the C⁵ H6 resonance, and a downfield shift of 0.17 ppm for the C¹⁹ H5 resonance.

Figure 5.

(A) Chemical shift differences of cytosine H5 and H6 protons of the OPdG-2BD duplex relative to the unmodified 2BD duplex. (B) Chemical shift differences of cytosine H5 and H6 protons of the M₁dG-2BD duplex relative to the unmodified 2BD duplex. The data for the cytosine H5 protons are colored gray and the data for the cytosine H6 protons black. (C) Chemical shift differences of nucleotide base protons A⁻² → G¹¹ of the OPdG-2BD duplex relative to the M₁dG-2BD duplex. (D) Chemical shift differences of nucleotide base protons C¹² → T²² of the OPdG-2BD duplex relative to the M₁dG-2BD duplex. The data for the nucleotide base aromatic H8/H6 protons are colored black, the data for the cytosine aromatic H5 protons gray, and the data for the sugar H1′ protons white. In all instances, Δδ = δ_{modified oligodeoxynucleotide} – δ_{unmodified oligodeoxynucleotide} (parts per million).

Chemical Shift Comparisons between the OPdG-2BD and M₁dG-2BD Duplexes

Panels C and D of Figure 5 show chemical shift differences for the pyrimidine H5 and H6 or purine H8 and deoxyribose H1′ protons, comparing the OPdG-2BD and M₁dG-2BD duplexes. The ¹H chemical shifts of the non-reiterated base pairs G⁶•C¹⁷, G⁷•C¹⁶, C⁸•G¹⁵, A⁹•T¹⁴, T¹⁰•A¹³, and G¹¹•C¹² were conserved in both duplexes. Significant chemical shift perturbations were observed for the reiterated base pairs associated with the two-nucleotide bulge, suggesting that the two-nucleotide bulge was differentially accommodated in the OPdG-2BD versus the M₁dG-2BD duplex. The biggest chemical shift difference between the OPdG-2BD duplex and the M₁dG-2BD duplex was 0.47 ppm, observed for the C⁵ H5 resonance. Other significant shift changes were observed in the modified strand, 0.33 ppm for the C¹ H1′ resonance, 0.23 ppm for the G² H1′ resonance, 0.18 ppm for the C³ H5 resonance, and 0.30 ppm for the C⁵ H6 resonance. In the complementary strand, the G¹⁸ H8 resonance exhibited a 0.26 ppm shift.

Equilibrium of the OPdG-2BD and M₁dG-2BD Duplexes

The initially formed OPdG-2BD and M₁dG-2BD duplexes were not at equilibrium. The conversion of the OPdG-2BD duplex toward an equilibrium mixture of the M₁dG-2BD and OPdG-2BD duplexes was monitored as a function of time, by monitoring the COSY spectrum. Figure 6 monitors the ratio of resonance intensities from the C¹ H5–H6 COSY cross-peak of either OPdG or M₁dG as a function of time, beginning with freshly prepared OPdG-2BD or M₁dG-2BD duplexes. This cross-peak reflected the environment of the C¹•G²⁰ base pair, located at the 5′-terminus of the iterated repeat sequence with respect to the OPdG-modified nucleotide X⁴. Over a period of 140 days at 25 °C, the eight COSY cross-peaks arising from the OPdG-2BD duplex decreased in intensity and eight new cross-peaks corresponding to formation of the M₁dG-2BD duplex appeared and grew in intensity. As noted above, for the OPdG-2BD duplex, the sequential connectivities in the reiterated region, T²² N3H → T⁻¹ N3H → G²⁰ N1H → G² N1H → G¹⁸ N1H → G⁴ N1H → G⁶ N1H, were disturbed. As the OPdG duplex shifted toward the M₁dG-2BD duplex, these sequential connectivities appeared in the spectrum. At equilibrium, the M₁dG-2BD duplex was slightly favored. The equilibrium chemistry of a freshly prepared M₁dG-2BD duplex was also monitored. At equilibrium, the COSY spectrum of the initially prepared M₁dG-2BD duplex was identical to that of the OPdG-2BD duplex at equilibrium.

Figure 6.

Intensity ratios of the two C¹ H5 → C¹ H6 cross-peaks in the COSY spectrum, arising from M₁dG and OPdG, as a function of time: (A) freshly prepared OpdG-2BD duplex and (B) freshly prepared M₁dG-2BD duplex. The solid lines represent the best fits through the data in both plots. The errors in measuring the ratios are estimated to be ±2%.

Discussion

Interest in the structure of the OPdG and M₁dG adducts embedded in the frameshift-prone hisD3052-iterated (CG)₃ repeat sequence arose from the observation that MDA induced frameshift mutations (32). The M₁dG and OPdG adducts are chemically distinct. One would predict that the biological processing of MDA- or base propenal-induced damage sites in DNA depends upon whether the damage exists as the exocyclic adduct M₁dG or as its OPdG rearrangement product. Site-specific mutagenesis experiments involving M₁dG (25) or its chemically stable analogue PdG (45), which cannot open to OPdG, indicated that PdG gave greater numbers of point mutations. Similarly, the acrolein-induced γ-hydroxyl-1,N²-PdG adduct, which also underwent ring opening (46), was found to not be miscoding in vivo (47, 48).

It has not been possible to examine the M₁dG adduct with respect to structure in fully complementary DNA duplexes due to the fact that when placed opposite dC, it rapidly rearranges to the OPdG adduct (28). The saturated analogue 1,N²-propanodG (PdG adduct) (49) was used by our laboratory (30, 50–54) as well as by other laboratories (55–58) as a stable structural surrogate for exocyclic 1,N²-dG adducts such as the M₁dG adduct and the acrolein γ-OH-PdG adduct. The PdG adduct reduced the thermal stability, transition enthalpy, and transition free energy of duplex DNA when positioned opposite dC or dA (59). When placed in the hisD3052 gene-iterated CG repeat sequence at pH 5.8, 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 (50). In a noniterated duplex at pH 5.2, only PdG and not its 3′-neighbor base pair rotated about the glycosyl bond into the syn conformation (54). Presumably, the syn conformation of the glycosyl bond relieves steric strain associated with incorporation of the 1,N²-dG annelation product, which cannot participate in Watson–Crick hydrogen bonding, into the DNA duplex. In contrast, structural studies of the OPdG adduct in the fully complementary iterated repeat contained in the hisD3052 oligodeoxynucleotide showed that it remained in the anti orientation about the glycosyl bond and oriented in the minor groove of the DNA duplex such that it did not interfere with Watson–Crick base pairing (31).

The iterated CG repeat contained in the hisD3052 oligodeoxynucleotide is thought to be prone to frameshifts by slippage of either the template or the primer strand during DNA replication (60). The hisD3052 mutation arose from the histidinol dehydrogenase gene of S. typhimurium by deletion of a cytosine induced by ICR-191 (61, 62). It is reversed by additions and deletions that restore the reading frame but do not necessarily reverse the forward mutation (63). The most common reversion is a CG deletion in the reiterated sequence (64–67). The unmodified 2BD duplex and the M₁dG-2BD (33) and OPdG-2BD duplexes model the intermediate structures leading to two-base deletions.

Bulge Migration in the Unmodified 2BD Duplex

Woodson and Crothers (35) first characterized the phenomenon of bulge migration within iterated DNA sequences. Several lines of evidence suggest that formation of a two-nucleotide bulge within the unmodified 2BD duplex was also accompanied by bulge migration. Significantly, a complete set of sequential NOE connectivities between nonexchangeable base aromatic and deoxyribose anomeric protons was observed (Figure 2 A,B). This observation was consistent with rapid migration of the two-nucleotide bulge in the hisD3052 sequence such that on the NMR time scale, a set of sequential NOEs consistent with a right-handed duplex is observed for nucleotides C¹, G², C³, G⁴, C⁵, and G⁶. The presence of a localized two-nucleotide bulge on the NMR time scale would have predicted a break in these NOE connectivities, which was observed for the M₁dG-2BD duplex (33). In that instance, for the modified strand, the C³ H1′ → M₁dG H2 sequential NOE (the imidazole proton of M₁dG, corresponding to G H8 in the unmodified nucleotide) was missing. In the complementary strand, the C¹⁷ H1′ → G¹⁸ H8 NOE was missing (33).

The observation that the eight cytosine H6–H5 COSY cross-peaks in Figure 1 A were well resolved corroborated the conclusion that the bulge migrated rapidly compared to the NMR time scale. In contrast, for the M₁dG-2BD duplex (Figure 1C), the localized bulge exhibited spectral line broadening adjacent to the M₁dG lesion, for the C³ and C⁵ cytosine H5 and cytosine H6 resonances, the 5′- and 3′-neighbors of the M₁dG lesion, respectively. In the complementary strand, line broadening was observed for C¹⁹ H5 and H6 (33).

The rapid bulge migration was also evident from the increased rate of solvent exchange of the dG N1H imino protons within the (CG)₃ iterated repeat sequence. In the non-reiterated portion of the bulged oligodeoxynucleotide, the dG N1H and dT N3H imino resonances arising from Watson–Crick base pairs G⁶•C¹⁷, G⁷•C¹⁶, C⁸•G¹⁵, A⁹•T¹⁴, and T¹⁰•A¹³ were observed in the fardownfield region of the ¹H NMR spectrum, indicating that these base pairs had long lifetimes with respect to exchange of the hydrogen-bonded protons with solvent. For these base pairs in the non-reiterated portion of the bulged oligodeoxynucleotide, the normal patterns of NOEs between the dG N1H and dC N⁴ exocyclic amino protons, characteristic of Watson–Crick base pairing, were observed in the ¹H NMR spectrum. In contrast, the dG N1H imino resonances arising from nucleotides G²⁰, G², G¹⁸, and G⁴ in the reiterated portion of the bulged oligodeoxynucleotide exhibited significant spectral line broadening associated with an increased rate of solvent exchange. However, for nucleotides C¹, C¹⁹, C³, and C⁵ in the reiterated portion of the bulged oligodeoxynucleotide, NOEs between the dC N⁴ exocyclic amino protons and the dG N1H protons of complementary deoxyguanosines, characteristic of individual Watson–Crick base pairs, could be assigned, indicating that transient Watson–Crick base pairing involving all four deoxycytosines in the reiterated portion of the duplex must be occurring. For the unmodified 2BD duplex, the T⁻¹ N3H imino proton resonance arising from the T⁻¹•A²¹ base pair adjacent to the iterated repeat was not observed. Also, no cross-peaks were observed between the exocyclic N⁴ amino protons of C¹ and guanine imino protons. In contrast, for the M₁dG-2BD duplex in which the two-nucleotide bulge was localized, an interruption in the sequential NOE connectivity between the dG N1H protons of adjacent base pairs was observed at G¹⁸, the base pair 5′ to the M₁dG lesion, and no NOE was observed between G¹⁸ N1H and G⁶ N1H, which localized the M₁dG adduct between base pairs C⁵•G¹⁸ and G⁶•C¹⁷ (33). For the M₁dG-2BD duplex, the T⁻¹ N3H imino proton resonance arising from the T⁻¹•A²¹ base pair adjacent to the iterated repeat was observed. Also, cross-peaks from the N⁴ exocyclic amine protons of C¹ to the G²⁰ N1H imino proton were observed, indicating the presence of the C¹•G²⁰ base pair (33).

Minor Groove Orientation of the OPdG Adduct in the OPdG-2BD Duplex

The pattern of NOEs observed between the OPdG moiety and the DNA in the OPdG-2BD duplex was consistent with a minor groove orientation of the OPdG adduct. The NOEs shown in Figure 4 involved X⁴ H1′, and C⁵ H1′, H4′, and H5′. These NOEs served to locate the OPdG moiety within the minor groove. Thus, the orientation of OPdG in the OPdG-2BD duplex was similar to that in the fully complementary hisD3052 oligodeoxynucleotide (31). However, the pattern of NOEs within the minor groove was different. In the fully complementary duplex, OPdG exhibited NOEs to minor groove protons in the complementary strand, e.g., C¹⁹ H1′, G²⁰ H1′, G²⁰ H4′, and G²⁰ H5′ and H5″ (31).

Bulge Migration in the OPdG-2BD Duplex

The spectra of the OPdG-2BD duplex were similar to those from the unmodified 2BD duplex and differed from the spectra obtained for the M₁dG-2BD duplex (33). These observations suggested that formation of a two-nucleotide bulge within the OPdG-2BD duplex was also accompanied by bulge migration. The observation of a complete set of sequential NOE connectivities between nonexchangeable base aromatic and deoxyribose anomeric protons (Figure 2 C, D) was consistent with rapid migration of the two-nucleotide bulge such that on the NMR time scale, a set of sequential NOEs consistent with a right-handed duplex was observed for nucleotides C¹, G², C³, X⁴, C⁵, and G⁶. Eight well-resolved cytosine H6–H5 COSY cross-peaks were observed (Figure 1 B), also corroborating the conclusion that the bulge migrated rapidly on the NMR time scale. In the non-reiterated portion of the OPdG-2BD duplex, the dG N1H and dT N3H imino resonances from Watson–Crick base pairs G⁶•C¹⁷, G⁷•C¹⁶, C⁸•G¹⁵, A⁹•T¹⁴, and T¹⁰•A¹³ were observed, indicating that these base pairs had long lifetimes with respect to exchange of the hydrogen-bonded protons with solvent. Furthermore, for these base pairs, the normal patterns of NOEs between the dG N1H and dC N⁴ exocyclic amino protons, characteristic of Watson–Crick base pairing, were observed. In contrast, the dG N1H imino resonances arising from nucleotides G²⁰, G², G¹⁸, and X⁴ in the reiterated portion of the OPdG-2BD duplex exhibited spectral line broadening. For these nucleotides, the sequential NOEs between the dG N1H protons of adjacent base pairs and the NOEs between the dG N1H protons and the dC N⁴ exocyclic amino protons, characteristic of individual Watson–Crick base pairs, could not be unequivocally assigned. In addition, the T⁻¹ N3H imino proton resonance arising from the T⁻¹•A²¹ base pair adjacent to the iterated repeat was not observed, indicating that it also underwent solvent exchange at an increased rate (Figure 3B). The differential pattern of minor groove NOEs that was observed between the OPdG-2BD duplex and the fully complementary hisD3052 duplex containing the OPdG adduct (31) was also consistent with bulge migration in the OPdG-2BD duplex. Thus, the rapid migration of the bulge precluded observation of NOEs to minor groove protons in the complementary strand but maintained the observation of NOEs to the 3′-neighbor nucleotide C⁵ in the modified strand of the duplex.

The rapid rate of bulge migration is consistent with the observation that the OPdG adduct facilitates Watson–Crick base pairing, which was confirmed by Riggins et al. (30). Because OPdG does not interfere with Watson–Crick hydrogen bonding and is located in the minor groove, the location of the 2BD bulge is not localized. In contrast, M₁dG does interfere with Watson–Crick hydrogen bonding, and its accommodation in duplex DNA presumably requires its reorientation into the syn conformation about the glycosyl bond, similar to the case with the PdG adduct. Accordingly, the localization of the two-nucleotide bulge in the M₁dG-2BD duplex, involving the modified nucleotide X⁴ and its 3′-neighbor C⁵ (33), is favored.

Chemical Shift Differences among the Unmodified 2BD Duplex, the OPdG-2BD Duplex, and the M₁dG-2BD Duplex

The notion that the OPdG and M₁dG adducts existed in different environments within the iterated repeat sequence containing the two-nucleotide bulge was corroborated by examination of chemical shift effects (Figure 5). Only modest differences were observed when the chemical shifts for the cytosine H5 and H6 resonances of the unmodified 2BD duplex were compared to those of the OPdG-2BD duplex, consistent with the idea that these two duplexes behaved similarly. In contrast, a comparison of the unmodified 2BD duplex with the M₁dG-2BD duplex showed a significant difference at nucleotide C5, the 3′-neighbor to the M₁dG-adducted nucleotide. Likewise, large chemical shift differences were observed when the OPdG duplex was compared with the M₁dG duplex. Significantly, these chemical shift differences were localized to the iterated repeat sections of the two bulged duplexes. Only minimal chemical shift differences were observed for the non-reiterated regions of the M₁dG-2BD duplex versus the OPdG-2BD duplex, consistent with the conclusion that the two bulged duplexes differed in the manners in which the respective adducts were accommodated within the iterated repeat sequence.

Differential Rates of Bulge Migration in the Bulged M₁dG-2BD and OPdG-2BD Duplexes Modulate the Rate at Which Equilibrium between M₁dG and OPdG Is Achieved

The differential rates of bulge migration in the M₁dG-2BD and OPdG-2BD duplexes affect the rate at which equilibrium between M₁dG and OPdG is achieved, as compared to that of fully complementary duplexes containing the M₁dG or OPdG adducts. For the fully complementary hisD3052 duplex, the M₁dG adduct placed opposite dC rearranged to OPdG rapidly; its conversion was completed in minutes. For the M₁dG-2BD duplex, the two-base bulge was localized and consisted of M₁dG and the 3′-neighbor (33). As such, the M₁dG-2BD bulge lacks a hydrated cytosine positioned to facilitate opening of M₁dG (33); thus, at neutral pH, ring opening of M₁dG to OPdG occurs slowly in the M₁dG-2BD duplex. This study indicates that at 25 °C, equilibrium between M₁dG and OPdG, favoring M₁dG (33), was attained over a period of 140 days (Figure 6). This suggests that the localized bulge in the M₁dG-2BD duplex undergoes a slow migration. This was not recognized in the earlier studies (33), which were conducted using a freshly prepared M₁dG-2BD duplex. A slow migration of the bulge in the M₁dG-2BD duplex would transiently relocate M₁dG to a position opposite from nucleotide C¹⁹ in the complementary strand, which would facilitate ring opening of the OPdG adduct, via the mechanism proposed by Riggins et al. (29). Once the OPdG adduct forms at neutral pH, its more rapid bulge migration rate then slows the rate of its reversion back to the localized bulge in the M₁dG-2BD duplex. We conclude that the step in the formation of M₁dG, believed to be the dehydration of the initially formed 8-hydroxy-6,7-propenodG intermediate (30), is slow compared to bulge migration. Hence, it requires many days for the equilibrium involving OPdG and M₁dG in the OPdG-2BD and M₁dG-2BD duplexes to be attained.

Comparison to Other Oligodeoxynucleotides Containing Bulged Nucleotides

Previous studies examined oligodeoxynucleotides containing unpaired bases or bulges of various sequence contexts and length (68–75). Unpaired purines generally adopt intrahelical conformations in solution (74, 76, 77). For unpaired pyrimidines, both intrahelical (7879)and extrahelical conformations (6980)have been observed. The role of flanking DNA sequences in determining the conformation of bulged pyrimidines was demonstrated by studies demonstrating that bulged pyrimidines embedded in A•T tracts adopt an extrahelical conformation (70, 81).

Structure–Activity Relationships

The M₁dG-induced frame-shift mutations observed in vivo in E. coli and in COS-7 cells, and associated with the d(CpG)₃ iterated repeat, include −2 bp deletions (26). These are consistent with the observation that frameshift mutations in the iterated CG repeat sequence of the hisD3052 gene are typically −2 base deletions. These suggest mechanisms whereby the modified guanine and adjacent cytosine undergo transient dislocation during replication bypass (60, 82). This work demonstrates rapid bulge migration within the iterated repeat sequence of the hisD3052 gene containing the OPdG adduct. The occurrence of transient dislocation during replication could occur either prior to insertion of a nucleotide opposite M₁dG or, alternatively, after nucleotide insertion and prior to extension (83). It could also involve dissociation or re-association with the replication complex.

In any case, relationships between DNA structure and dynamics, and the formation and accommodation of transient dislocation complexes during DNA replication, are anticipated to be polymerase-specific (84). Both M₁dG and OPdG block replication by the Klenow fragment of DNA polymerase I, with M₁dG being approximately 6-fold more blocking than OPdG (85). DNA polymerase β induces two-base deletions when replicating past M₁dG in this iterated d(CpG)₃ sequence derived from the S. typhimuium hisD3052 gene. ² However, the interactions of M₁dG and OPdG with various Y-family polymerases (86, 87) remain to be determined and are likely to be crucial for understanding the mechanisms by which these lesions induce both frameshift and base pair substitution mutations in human cells. Significantly, structures of the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) have been obtained for binary and ternary complexes with primer templates site-specifically modified with 1,N²-ethenodeoxyguanosine (1,N²-εdG) (88, 89), an adduct that is structurally similar to M₁dG. The Dpo4 structures with the 1,N²-εdG adduct suggest that it uses several mechanisms, including a variation of dNTP-stabilized misalignment, to generate frameshifts when it encounters the exocyclic DNA adduct (88).