Structure of the 1,N(2)-etheno-2'-deoxyguanosine lesion in the 3'-G(epsilon dG)T-5' sequence opposite a one-base deletion

Abstract

The structure of the 1,N(2)-ethenodeoxyguanosine lesion (1,N(2)-epsilondG) has been characterized in 5'-d(CGCATXGAATCC)-3'.5'-d(GGATTCATGCG)-3' (X = 1,N(2)-epsilondG), in which there is no dC opposite the lesion. This duplex (named the 1-BD duplex) models the product of translesion bypass of 1,N(2)-epsilondG by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) [Zang, H., Goodenough, A. K., Choi, J. Y., Irimia, A., Loukachevitch, L. V., Kozekov, I. D., Angel, K. C., Rizzo, C. J., Egli, M., and Guengerich, F. P. (2005) J. Biol. Chem. 280, 29750-29764], leading to a one-base deletion. The T(m) of this duplex is 6 degrees C higher than that of the duplex in which dC is present opposite the 1,N(2)-epsilondG lesion and 8 degrees C higher than that of the unmodified 1-BD duplex. Analysis of NOEs between the 1,N(2)-epsilondG imidazole and deoxyribose H1' protons and between the 1,N(2)-epsilondG etheno H6 and H7 protons and DNA protons establishes that 1,N(2)-epsilondG adopts the anti conformation about the glycosyl bond and that the etheno moiety is accommodated within the helix. The resonances of the 1,N(2)-epsilondG H6 and H7 etheno protons shift upfield relative to the monomer 1,N(2)-epsilondG, attributed to ring current shielding, consistent with their intrahelical location. NMR data reveal that Watson-Crick base pairing is maintained at both the 5' and 3' neighbor base pairs. The structure of the 1-BD duplex has been refined using molecular dynamics calculations restrained by NMR-derived distance and dihedral angle restraints. The increased stability of the 1,N(2)-epsilondG lesion in the absence of the complementary dC correlates with the one-base deletion extension product observed during the bypass of the 1,N(2)-epsilondG lesion by the Dpo4 polymerase, suggesting that stabilization of this bulged intermediate may be significant with regard to the biological processing of the lesion.

Scheme 1. (A) 1-BD-Modified Oligodeoxynucleotide Numbering Scheme and (B) Structure and Numbering Scheme for the 1,N²-εdG Adduct

The imidazole proton of the 1,N²-εdG nucleotide is designated as H2, corresponding to the H8 proton in guanine.

Figure 1

Temperature-dependent ¹H NMR spectra (imino proton region) for the 1-BD (A) and unmodified 1-BD (B) duplex.

Figure 2

Expanded plot of a phase sensitive NOESY spectrum (250 ms) in D₂O buffer for the 1-BD oligodeoxynucleotide duplex showing the sequential NOEs from the aromatic to H1′ protons. The spectrum was recorded at 25 °C. (A) Connectivities are traced for the 1,N²-εdG-modified strand: Additional cross-peaks, (A) A⁴ H2 → A⁴ H1′, (B) A⁴ H2 → T⁵ H1′, (C) A⁸ H2 → A⁸ H1′, (D) A⁸ H2 → A⁹ H1′, (E) A⁹ H2 → A⁹ H1′, (F) A⁹ H2 → T¹⁰ H1′, (G) G² H8 → C³ H5, (H) T¹⁰ H6 → C¹¹ H5, and (I) C¹¹ H6 → C¹² H5. (B) Connectivities are traced for the complementary strand: Additional cross-peaks, (J) A¹⁵ H2 → A¹⁵ H1′, (K) A¹⁵ H2 → T¹⁶ H1′, (L) A¹⁹ H2 → A¹⁹ H1′, (M) A¹⁹ H2 → T²⁰ H1′, (N) T¹⁷ H6 → C¹⁸ H5, and (O) G²¹ H8 → C²² H5.

Figure 3

(A) Downfield region of the ¹H NMR spectrum for the 1-BD duplex showing the imino proton resonances. (B) Expanded plot of a NOESY spectrum with a mixing time of 250 ms for the 1-BD duplex showing the sequential NOEs between the imino protons and the amino and base protons: (A and A′) G² N1H → C²² N⁴H, h/n; (B and B′) G²¹ N1H → C³ N⁴H, h/n; (C and C′) G⁷ N1H → C¹⁸ N⁴H, h/n; (D and D′) G¹⁴ N1H → C¹¹ N⁴H, h/n; (E) T²⁰ N3H → A⁴ H2; (F) T⁵ N3H → A¹⁹ H2; (G) T¹⁷ N3H → A⁸ H2; (H) T¹⁶ N3H → A⁹ H2; and (I) T¹⁰ N3H → A¹⁵ H2. The symbols h and n denote hydrogen-bonded and non-hydrogen-bonded amino protons of cytosine, respectively. (C). Expanded plot of a NOESY spectrum showing sequential NOE connectivity for the imino protons of the G²·C²³ → G¹⁴·C¹¹ base pairs for the 1-BD duplex. The labels designate the imino proton of the indicated nucleotide. The spectra were recorded at 7 °C.

Figure 4

Expanded region of the ¹H COSY spectrum showing a cross-peak between etheno H7 and H6 protons. The spectrum was recorded at 25 °C.

Figure 5

(A) Time-dependent ¹H NMR spectra showing H−D exchange of the H7 etheno proton at 50 °C. The NMR spectra were recorded at 25 °C: (a) immediately after the sample was dissolved in buffer, (b) after 96 h, (c) after 216 h, and (d) after 576 h. (B) Change in the integrated intensity of H7 (5.84 ppm) and H6 (5.55 ppm) proton resonances with time. ^#The cytosine (C²³) H5 proton resonance was taken as a reference.

Figure 6

¹H NOESY spectrum (tile plot) showing the assignment of exocyclic etheno protons H7 and H6 and NOEs between the etheno and DNA protons. The NOESY spectrum was recorded at 25 °C with a mixing time of 250 ms: (a) X⁶ H7 → X⁶ H6, (b) X⁶ H7 → C¹⁸ H2′, (c) X⁶ H7 → C¹⁸ H2′′, (d) X⁶ H7 → A¹⁹ H5′/H5′′, (e) X⁶ H7 → C¹⁸ H5, (f) X⁶ H7 → C¹⁸ H1′, (g) X⁶ H7 → C¹⁸ H6, (h) X⁶ H7 → A¹⁹ H2, (i) X⁶ H6 → C¹⁸ H2′, (j) X⁶ H6 → C¹⁸ H2′′, (k) X⁶ H6 → A¹⁹ H5′/H5′′, (l) X⁶ H6 → A¹⁹ H5′/H5′′, (m) X⁶ H6 → A¹⁹ H4′, (n) X⁶ H6 → C¹⁸ H1′, (o) X⁶ H6 → A¹⁹ H1′, (p) X⁶ H6 → A¹⁹ H2, (q) X⁶ H6 → C¹⁸ H6, and (r) X⁶ H6 → A¹⁹ H8.

Figure 7

¹H NOESY spectrum (tile plot) showing the NOEs between etheno protons and imino protons from neighboring base pairs. The NOESY spectrum was recorded at 7 °C with a mixing time of 250 ms.

Figure 8

Chemical shift differences observed for the 1-BD duplex when compared to those of the corresponding unmodified 1-BD duplex.

Figure 9

View of the central 5′-TXG-3′ segment of the 1-BD duplex as seen from the major groove.

Figure 10

View from the top of the helix showing stacking interactions on the central segment of the duplex. (A) Interaction between the T⁵·A¹⁹ and X⁶ base pairs. (B) Interaction between the X⁶ and G⁷·C¹⁸ base pairs.