DNA sequence modulates the conformation of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline in the recognition sequence of the NarI restriction enzyme

Abstract

The conformations of C8-dG adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) positioned in the C-X1-G, G-X2-C, and C-X3-C contexts in the C-G1-G2-C-G3-C-C recognition sequence of the NarI restriction enzyme were compared, using the oligodeoxynucleotides 5'-d(CTCXGCGCCATC)-3'.5'-d(GATGGCGCCGAG)-3', 5'-d(CTCGXCGCCATC)-3'.5'-d(GATGGCGCCGAG)-3', and 5'-d(CTCGGCXCCATC)-3'.5'-d(GATGGCGCCGAG)-3' (X is the C8-dG adduct of IQ). These were the NarIIQ1, NarIIQ2, and NarIIQ3 duplexes, respectively. In each instance, the glycosyl torsion angle chi for the IQ-modified dG was in the syn conformation. The orientations of the IQ moieties were dependent upon the conformations of torsion angles alpha' [N9-C8-N(IQ)-C2(IQ)] and beta' [C8-N(IQ)-C2(IQ)-N3(IQ)], which were monitored by the patterns of 1H NOEs between the IQ moieties and the DNA in the three sequence contexts. The conformational states of IQ torsion angles alpha' and beta' were predicted from the refined structures of the three adducts obtained from restrained molecular dynamics calculations, utilizing simulated annealing protocols. For the NarIIQ1 and NarIIQ2 duplexes, the alpha' torsion angles were predicted to be -176 +/- 8 degrees and -160 +/- 8 degrees , respectively, whereas for the NarIIQ3 duplex, torsion angle alpha' was predicted to be 159 +/- 7 degrees . Likewise, for the NarIIQ1 and NarIIQ2 duplexes, the beta' torsion angles were predicted to be -152 +/- 8 degrees and -164 +/- 7 degrees , respectively, whereas for the NarIIQ3 duplex, torsion angle beta' was predicted to be -23 +/- 8 degrees . Consequently, the conformations of the IQ adduct in the NarIIQ1 and NarIIQ2 duplexes were similar, with the IQ methyl protons and IQ H4 and H5 protons facing outward in the minor groove, whereas in the NarIIQ3 duplex, the IQ methyl protons and the IQ H4 and H5 protons faced into the DNA duplex, facilitating the base-displaced intercalated orientation of the IQ moiety [Wang, F., Elmquist, C. E., Stover, J. S., Rizzo, C. J., and Stone, M. P. (2006) J. Am. Chem. Soc. 128, 10085-10095]. In contrast, for the NarIIQ1 and NarIIQ2 duplexes, the IQ moiety remained in the minor groove. These sequence-dependent differences suggest that base-displaced intercalation of the IQ adduct is favored when both the 5'- and 3'-flanking nucleotides in the complementary strand are guanines. These conformational differences may correlate with sequence-dependent differences in translesion replication.

Chart 1

Metabolic Activation of IQ

Chart 2

(A) C8-dG IQ Adducted NarIIQ1 Dodecamer, (B) C8-dG IQ Adducted NarIIQ2 Dodecamer, (C) C8-dG IQ Adducted NarIIQ3 Dodecamer, and (D) C8-dG IQ Adduct^a

Figure 1

Expanded plots from the aromatic proton–anomeric proton region of the 800.13 MHz NOESY spectrum for the modified NarIIQ1 and NarIIQ2 duplexes at 15 °C using a mixing time of 250 ms, showing sequential NOE connectivity. (A) Nucleotides C¹–C¹² of the NarIIQ1 duplex. (B) Nucleotides G¹³–G²⁴ of the NarIIQ1 duplex. (C) Nucleotides C¹–C¹² of the modified strand in the NarIIQ2 duplex. (D) Nucleotides G¹³–G²⁴ of the complementary strand in the NarIIQ2 duplex.

Figure 2

Comparison of expanded plots of the imino proton region of the ¹H NOESY spectra for (A) the NarIIQ1 and (B) NarIIQ2 duplexes. In the bottom panels are expanded plots showing sequential NOE connectivity for the imino protons of base pairs T²·A²³ to T¹¹·A¹⁴ at 15 °C. The labels represent the imino proton of the designated base. In the middle panels are NOE connectivities between the imino protons and the base amino protons. The NOE cross-peaks involving the imino protons are labeled in the figure as follows: (A) (a′ and a) G²² N1H → C³ NH₂-4b,e and (b′ and b) G⁵ N1H → C²⁰ NH₂-4b,e and (B) (a′ and a) G⁴ N1H → C²¹ NH₂-4b,e and (b′ and b) G¹⁹ N1H → C⁶ NH₂-4b,e. In the top panels are NOE connectivities between the imino protons and the IQ methyl protons. The IQ-DNA cross-peaks are labeled as follows: (A) 1, G²² N1H → X⁴ CH₃; and 2, G⁵ N1H → X⁴ CH₃; and (B) 1, G⁴ N1H → X⁵ CH₃; and 2, G¹⁹ N1H → X⁵ CH₃.

Figure 3

Assignments of the IQ proton resonances. Expanded plots from (A) the COSY spectrum and (B) the aromatic–aromatic region of the NOESY spectrum at 15 °C for the IQ-adducted NarIIQ1 duplex. Expanded plots from (C) the COSY spectrum and (D) the aromatic–aromatic region of the NOESY spectrum at 15 °C for the IQ-adducted NarIIQ2 duplex.

Figure 4

Tile plots showing NOE cross-peaks between nonexchangeable protons of DNA and IQ protons in the NarIIQ1 and NarIIQ2 duplexes. (A) NOE cross-peaks for the NarIIQ1 duplex: (a) C²⁰ H2′ → IQ H9A, (b) C²⁰ H2″ → IQ H9A, (c) G²² H5′ → IQ H9A, (d) C²⁰ H3′ → IQ H9A, (e) C²¹ H4′ → IQ H9A, (f) C²¹ H1′ → IQ H9A, (g) C²⁰ H1′ → IQ H9A, (h) C²⁰ H8 → IQ H9A, (i) G¹⁷ H8 → IQ H9A, (j) G²² H1′ → IQ H4A, (k) G²² H1′ → IQ H5A, (l) X⁴ H1′ → IQ CH₃, (m) G⁵ H1′ → IQ CH , (n) G⁵ H5′ → IQ CH₃, (o) G²² N1H → IQ CH₃, and (p) G⁵ N1H → IQ CH₃. (B) NOE cross-peaks for the NarIIQ2 duplex: (a) G¹⁹ H2″ → IQ H9A, (b) G¹⁹ H3′ → IQ H9A, (c) G¹⁹ H1′ → IQ H9A, (d) C²⁰ H1′ → IQ H9A, (e) C²¹ H6 → IQ H9A, (f) G¹⁹ H8 → IQ H9A, (g) C²¹ H1′ → IQ H4A, (h) C²⁰ H2′ → IQ H5A, (i) C²⁰ H2″ → IQ H5A, (j) G¹⁹ H2″ → IQ H8A, (k) C²¹ H4′ → IQ H5A, (l) C²¹ H5 → IQ H5A, (m) C²¹ H1′ → IQ H5A, (n) G¹⁹ H8 → IQ H8A, (o) X⁵ H1′ → IQ CH₃, (p) C⁶ H1′ → IQ CH₃, (q) C²¹ H1′ → IQ CH₃, (r) C⁶ H4′ → IQ CH₃, (s) G⁴ N1H → IQ CH₃, and (t) G¹⁹ N1H → IQ CH₃.

Figure 5

Expanded COSY contour plot at 15 °C establishing the connectivity between the H1′ and H2′ and H2″ protons. (A) NarIIQ1 duplex. The H2′ and H2″ protons of nucleotides C³, X⁴, G⁵, C²⁰, C²¹, and G²² adjacent to the lesion site are connected by lines and labeled. (B) NarIIQ2 duplex. The H2′ and H2″ protons of nucleotides G⁴, X⁵, C⁶, G¹⁹, C²⁰, and C²¹ adjacent to the lesion site are connected by lines and labeled.

Figure 6

Chemical shift changes of (A) aromatic protons H6 and H8 and (B) anomeric H1′ protons of the NarIIQ1 duplex and (C) aromatic protons H6 and H8 and (D) anomeric H1′ protons of the NarIIQ2 duplex, relative to the unmodified duplex, where Δδ = δ_{modified oligodeoxynucleotide} – δ_{unmodified oligodeoxynucleotide} (parts per million).

Figure 7

Stereoviews of 10 randomly seeded superimposed structures of the (A) NarIIQ1 and (B) NarIIQ2 duplexes emergent from rMD simulated annealing calculations, looking into the minor groove at the lesion site.

Figure 8

Distribution of R^x₁ values calculated using CORMA (72). (A) Nucleotides C¹–C¹² of the modified NarIIQ1 duplex. (B) Nucleotides G¹³–G²⁴ of the modified NarIIQ1 duplex. (C) Nucleotides C¹–C¹² of the modified NarIIQ2 duplex. (D) Nucleotides G¹³–G²⁴ of the modified NarIIQ2 duplex. The black bars represent intranucleotide values and the gray bars internucleotide values.

Figure 9

Comparison of averaged refined structures, looking into the minor groove, and normal to the helix axis of the central segment: (A) NarIIQ1 duplex and (B) NarIIQ2 duplex. The NOEs defining the IQ orientation are indicated by the green dashed lines.

Figure 10

Average refined structure of the NarIIQ3 duplex, looking into the major groove, and normal to the helix axis of the central segment (63). The NOEs defining the IQ orientation are indicated by the green dashed lines.

Figure 11

Base stacking orientations of the NarI, NarIIQ1, NarIIQ2, and NarIIQ3 (63) duplexes. (A) Unmodified duplex detailing base stacking corresponding to the modified duplexes. (B) NarIIQ1, NarIIQ2, and NarIIQ3 (63) duplexes detailing base stacking between the base pair at the modified position and its 5′ neighboring base pair, with the 5′ neighboring base pair aligned as in the unmodified duplex.

Figure 12

Base stacking orientations of the NarI, NarIIQ1, NarIIQ2, and NarIIQ3 (63) duplexes. (A) Unmodified duplex detailing base stacking corresponding to the modified duplexes. (B) NarIIQ1, NarIIQ2, and NarIIQ3 (63) duplexes detailing base stacking between the base pair at the modified position and its 3′ neighboring base pair, with the 3′ neighboring base pair aligned as in the unmodified duplex.