Base-Displaced Intercalated Structure of the N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone DNA Adduct

Abstract

3-Nitrobenzanthrone (3-NBA), an environmental mutagen found in diesel exhaust and a suspected carcinogen, undergoes metabolic reduction followed by reaction with DNA to form aminobenzanthrone (ABA) adducts, with the major alkylation product being N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). Site-specific synthesis of the C8-dG-ABA adduct in the oligodeoxynucleotide 5'-d(GTGCXTGTTTGT)-3':5'-d(ACAAACACGCAC)-3'; X = C8-dG-ABA adduct, including codons 272-275 of the p53 gene, has allowed for investigation into the structural and thermodynamic properties of this adduct. The conformation of the C8-dG-ABA adduct was determined using NMR spectroscopy and was refined using molecular dynamics (MD) calculations restrained by experimentally determined interproton distance restraints obtained from NOE experiments. The refined structure revealed that the C8-dG-ABA adduct formed a base-displaced intercalated conformation. The adducted guanine was shifted into the syn conformation about the glycosidic bond. The 5'- and 3'-neighboring base pairs remained intact. While this facilitated π-stacking interactions between the ABA moiety and neighboring bases, the thermal melting temperature (Tm) of the adduct-containing duplex showed a decrease of 11 °C as compared to the corresponding unmodified oligodeoxynucleotide duplex. Overall, in this sequence, the base-displaced intercalated conformation of the C8-dG-ABA lesion bears similarity to structures of other arylamine C8-dG adducts. However, in this sequence, the base-displaced intercalated conformation for the C8-dG-ABA adduct differs from the conformation of the N(2)-dG-ABA adduct reported by de los Santos and co-workers, in which it is oriented in the minor groove toward the 5' end of the duplex, with the modified guanine remaining in the anti conformation about the glyosidic torsion angle, and the complementary base remaining within the duplex. The results are discussed in relationship to differences between the C8-dG-ABA and N(2)-dG-ABA adducts with respect to susceptibility to nucleotide excision repair (NER).

Figure 1

Expanded plot of a NOESY spectrum of the C8-dG-ABA modified duplex, showing the sequential NOE connectivity between aromatic H8/H6 protons and deoxyribose H1' protons. A. The modified strand, showing bases G¹ through T¹². The NOE connectivity is broken at the C⁴ H1´-H6 cross peak and reinitiates at the X⁵ H1’ - T⁶ H6 cross peak since the modified X⁵ base does not have an H8 proton. B. The complementary strand connectivity showing assignments for bases A¹³ through C²⁴. The connectivity is broken at the A¹⁹ H1´→A¹⁹ H8 NOE and reinitiates at the C²⁰ H1´→C²⁰ H6 NOE; no NOE cross peak is observed between A¹⁹ H1´ and C²⁰ H6. The 900 MHz spectrum was acquired at 25 ºC using a 250 ms mixing time.

Figure 2

Expanded plots of the imino and amino regions of a NOESY spectrum of the C8-dG-ABA modified duplex. Left Panel: The imino proton region of the spectrum, showing NOEs between guanine N1H and thymine N3H imino protons. The broadening of the T⁶ N3H imino proton resonance is evident, Right Panel: NOEs between the guanine N1H and cytosine N⁴H amino protons and between thymine N3H and adenine H2 protons. The NOEs are assigned as follows: a1, G²¹ N1H → C⁴ N⁴H_b; a2, G²¹ N1H → C⁴ N⁴H_a; b1, G⁷ N1H → C¹⁸ N⁴H_b; b2, G⁷ N1H → C¹⁸ N⁴H_a; c1, G³ N1H → C²² N⁴H_b; c2, G³ N1H →C²² N⁴H_a; d1, G¹¹ N1H → C¹⁴ N⁴H_b; d2, G¹¹ N1H → C¹⁴ N⁴H_a; e1, T⁶ N3H → A¹⁹ H2; f1, T² N3H → A²³ H2; g1, T⁸ N3H → A¹⁷ H2; h1, T¹⁰ N3H → A¹⁵ H2; i1, T⁹ N3H → A¹⁶ H2. Note that cross-peak e1, arising from base pair T⁶:A¹⁹, the 3'-neighbor with respect to the modified base pair X⁵:C²⁰, is weaker than the cross peaks f1, g1, h1, and i1. The 900 MHz spectrum was acquired at 15 ºC using a 250 ms mixing time.

Figure 3

Expanded plots of regions of the NOESY spectrum of the C8-dG-ABA modified duplex, showing NOEs associated with the ABA cross-peaks (Chart 1). The boxed cross-peaks represent cross-peaks that are simultaneously observed in COSY spectra. In all cases, the cross-peaks are labeled to indicate proton resonances associated with the vertical (t1) axis first and proton resonances associated with the horizontal (t2) axis second. The 900 MHz spectrum was acquired at 15 º C using a 250 ms mixing time.

Figure 4

Expanded plots of a NOESY spectrum of the C8-dG-ABA modified duplex showing NOEs between adduct protons (Chart 1) and base and deoxyribose protons. The ABA H2, H4, and H5 protons presented NOEs to protons of the modified stand of the duplex. The ABA H8, H9, and H10 protons presented NOEs to the complementary strand of the duplex. The ABA H1 and H11 protons displayed no NOEs with either strand of the duplex. The cross-peaks are assigned as follows: a, ABA H6 → G²¹ H1´; b, ABA H2 → T⁶ CH₃; c, ABA H2 → C⁴ H5; d, ABA H4 → T⁶ CH₃, e, ABA H4 → X⁵ H2´´; f, ABA H4 → X⁵ H4´; g, ABA H4 → T⁶ H1´; h, ABA H4 → X⁵ H1´; i, ABA H5 → T⁶ H1´; j, ABA H5 → X⁵ H1´; k, ABA H8 → A¹⁹ H2´´; l, ABA H8 → A¹⁹ H2´; m, ABA H8 → A¹⁹ H1´; n, ABA H8 → C²⁰ H6; o, ABA H10 → A¹⁹ H2´´; p, ABA H10 → A¹⁹ H2´; q, ABA H10 → A¹⁹ H1´; r, ABA H9 → A¹⁹ H2´´; s, ABA H9 → A¹⁹ H2´; t, ABA H9 → C²⁰ H5´; u, ABA H9 → C²⁰ H5´´; v, ABA H9 → C²⁰ H4´; w, ABA H9 → A¹⁹ H3´; x, ABA H9 → A¹⁹ H1´; y, ABA H9 → A¹⁹ H8. The 900 MHz spectrum was acquired at 15 º C using a 250 ms mixing time.

Figure 5

Changes in chemical shifts for the protons of the ABA modified and surrounding nucleotides of the C8-dG-ABA modified duplex as compared to the unmodified duplex. Top Panel: Nucleotides of the modified strand, showing nucleotides C⁴ through T⁶. Bottom Panel: Nucleuotides of the complementary strand showing nucleotides A¹⁹ through G²¹. The base aromatic H6 or H8 protons are shown in white. The deoxyribose H1´ protons are shown in dots, the H2´ protons are shown in diagonal lines, the H2´´ protons are shown in horizontal lines, and the H3´ protons are shown in gray. The ?δ (ppm) values are calculated as δ_{unmodified duplex}- δ_{modified duplex}. Positive values of Δδ represent upfield shifts and negative values of Δδ represent downfield shifts, with respect to the unmodified duplex.

Figure 6

Overlay of ten lowest energy violation structures resulting from rMD calculations of the C8-dG-ABA modified duplex carried out using a simulated annealing protocol and using NOE generated distance restraints. The view is looking into the major groove.

Figure 7

Calculation of sixth root residual values (R₁^x) between theoretical NOEs predicted by complete relaxation matrix calculations and experimental NOEs for the averaged refined structure of the C8-dG-ABA duplex emergent from the rMD calculations, using the program CORMA. A. The intranucleotide, internucleotide and nucleotide to lesion R^X₁ values for individual nucleotides in the modified strand. B. The intranucleotide, internucleotide and nucleotide to lesion R^X₁ values for individual nucleotides in the complementary strand. The intranucleotide residuals are shaded in white. At the modified nucleotide X⁵, the intranucleotide residuals calculated for the C8-dG-ABA adduct are included with those of the X⁵ nucleotide. The DNA internucleotide residuals are shaded in gray. With the exception of the modified nucleotide X⁵, they indicate NOEs to the respective 3´-neighbor nucleotides. The internucleotide residuals between the C8-dG-ABA adduct and other nucleotides are shaded in dark gray. These involve nucleotides C⁴, T⁶, A¹⁹, C²⁰, and G²¹. In all cases, the sixth root residual factor was calculated as R₁^x = ∑[((I_o)_i^1/6)-((I_c)_i^1/6)/∑((I_o)_i^1/6)], in which I_c are NOE intensities calculated by complete relaxation matrix analysis of the refined structure and I_o are experimental NOE intensities.

Figure 8

Conformation of the C8-dG-ABA adduct as seen in the lowest violation structure emergent from rMD calculations. A. Base pairs C⁴:G²¹, X⁵:C²⁰, and T⁶:A¹⁹ as seen from the major groove. B. Base pairs C⁴:G²¹, X⁵:C²⁰, and T⁶:A¹⁹ as seen from the minor groove. The X⁵ base is rotated into the syn conformation at the glycosidic torsion angle; the C8-dG-ABA moiety is intercalated between base pairs C⁴:G²¹ and T⁶:A¹⁹; the complementary base C²⁰ is displaced into the major groove. The C⁴ and G²¹ base carbon atoms are in cyan. The X⁵ and C²⁰ base carbon atoms are in green. The T⁶ and A¹⁹ base carbon atoms are in orange. The adduct oxygen atom is shown in red and the nitrogen atom at the point of adduct attachment is shown in blue.

Figure 9

Base stacking interactions of the C8-dG-ABA adduct as seen in the lowest violation structure emergent from rMD calculations. A. View looking through the duplex from the 3´-side of the ABA adduct showing carbon atoms of bases X⁵ and C²⁰ in green and 3´-neighbor bases A¹⁹ and T⁶ in orange. B. View looking through the duplex from the 3´-side of the adducted nucleotide showing carbon atoms of bases of X⁵ and C²⁰ in green and 5'-neighbor bases G²¹ and C⁴ in cyan. The oxygen atom of the adduct is shown in red and the nitrogen at the point of adduct attachment atoms is shown in blue.

Chart 1

A. Chemical structure of the C8-dG-ABA adduct including numbering scheme of the adduct protons for NMR. B. The oligodeoxynucleotide sequence used in this work, showing numbering of the individual nucleotides.

Chart 2

Activation of 3-NBA into reactive electrophiles. Phase I activation occurs through nitroreduction. Phase II activation can occur through acetylation or sulfonation of the hydroxyl group (acetylation shown). Final activation occurs through solvolysis to form a nitrenium ion.

Chart 3

Reaction of the electrophilic nitrenium ion of 3-NBA with DNA to form three DNA aminobenzanthrone adducts. These occur at the C8 position of guanine, or the N² position of guanine and the N⁶ position of adenine to produce the three adducts shown.