Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA

Abstract

Endogenous 5-methylcytosine ((Me)C) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational 'hotspots' for smoking induced lung cancer. (Me)C enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5'-CCCGGCACCC GC[(15)N(3),(13)C(1)-G]TCCGCG-3', + strand) were prepared containing [(15)N(3), (13)C(1)]-guanine opposite unsubstituted cytosine, (Me)C, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2'-deoxynucleosides, N(2)-BPDE-dG adducts formed at the [(15)N(3), (13)C(1)]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N(2)-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE-DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N(2) position of guanine.

Scheme 1.

Metabolic activation of benzo[a]pyrene to BPDE and the formation of guanine adducts.

Scheme 2.

Nucleoside analogs employed in the present study.

Figure 1.

HPLC-ESI-MS/MS analysis of N²-BPDE-dG diastereomers formed at the ¹⁵N₃, ¹³C₁-labeled guanine when base paired with cytosine (A), 5-methylcytosine (B), 5-fluorocyrosine (C) or perimidin-2-one nucleoside (D) following treatment with (±)-anti-BPDE. Selected reaction monitoring of ¹⁵N₅,¹³C₁-N²-BPDE-dG was conducted by following the transitions m/z 574.1 [M+H]⁺→ 459.1 [M+2H-dR]⁺.

Scheme 3.

Experimental scheme for probing the reactivity of structurally modified C:G base pairs towards BPDE.

Figure 2.

Influence of alkyl substituents on cytosine on the yields of N²-BPDE-dG adducts at the base paired guanine. Synthetic DNA duplexes derived from p53 exon 5 [5′-CCCGGCA CCCGC[¹⁵N₃, ¹³C₁-G]TCCGCG-3′, (+) strand] containing cytosine, 5-methylcytosine, 5-ethylcytosine, 5-propylcytosine, or N⁴-ethylcytosine opposite ¹⁵N₃, ¹³C₁-G were treated with (±)-anti-BPDE (N = 4) (A) or (−)-anti-BPDE (N = 6) (B) and the extent of N²-BPDE-dG adduct formation at the isotopically tagged guanine was determined by HPLC-ESI⁺-MS/MS. Insert: pie charts showing the relative contributions of (−)-trans-N²-BPDE-dG, (+)-cis-N²-BPDE-dG, (−)-cis-N²- BPDE-dG and (+)-trans-N²-BPDE-dG to the total adduct number.

Figure 3.

Effects of cytosine methylation on the kinetics of formation of N²-BPDE-dG adducts at the base paired guanine (A) and at guanine bases elsewhere in the sequence (B). Increasing amounts of double-stranded DNA duplexes derived from p53 exon 5 (5′-CCCGGCACCCGC[¹⁵N₃, ¹³C₁-G]TCCGCG-3′, (+) strand) containing a single 5-methylcytosine opposite ¹⁵N₃, ¹³C₁-G were incubated with (±)-anti-BPDE (N = 3) for 1 min and quenched with 2-mercaptoethanol. The extent of N²-BPDE-dG adduct formation at the isotopically tagged guanine (¹⁵N₃, ¹³C₁-G) and at unlabeled guanines elsewhere in the sequence was quantified by HPLC-ESI⁺-MS/MS.

Figure 4.

Influence of C-5 halogen substituents on cytosine on the yields of (±)-anti-BPDE or (−)-anti-BPDE-induced N²-BPDE-dG adducts at the base paired guanine. Synthetic DNA duplexes derived from p53 exon 5 (5′-CCCGGCACCCGC[¹⁵N₃, ¹³C₁-G]TCCGCG-3′) containing cytosine, 5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, or 5-iodocytosine opposite the target G were treated with (±)-anti-BPDE (N = 4) (A) or (−)-anti-BPDE (N = 6) (B) and the extent of N²-BPDE-dG adduct formation at the isotopically tagged guanine was determined by HPLC-ESI⁺-MS/MS. Insert: pie charts showing relative contributions of (−)-trans-N²-BPDE-dG, (+)-cis-N²-BPDE-dG, (−)-cis-N²- BPDE-dG and (+)-trans-N²-BPDE-dG to the total adduct number. * = significantly different (P < 0.05).

Figure 5.

Influence of cytosine analogs with extended aromatic systems on the yields of N²-BPDE-dG adducts at the base paired guanine. Double-stranded DNA duplexes derived from p53 exon 5 (5′-CCCGGCACCCGC[¹⁵N₃, ¹³C₁-G]TCCGCG-3′) containing cytosine, 5-propynylcytosine (Pr-C), pyrrolocytosine (pyrrolo-C) or 6-phenylpyrrolocytosine (phenylpyrrolo-C) opposite the target G were treated with (±)-anti-BPDE (N = 4) (A) or (−)-anti-BPDE (N = 6) (B) and the extent of N²-BPDE-dG adduct formation at the isotopically tagged guanine was determined by HPLC-ESI⁺-MS/MS. Insert: pie charts showing relative contributions of (−)-trans-N²-BPDE-dG, (+)-cis-N²-BPDE-dG, (−)-cis-N²-BPDE-dG and (+)-trans-N²-BPDE-dG isomers. * = significantly different (P < 0.05).

Figure 6.

N²-BPDE-dG adduct formation at guanine bases placed in a DNA duplex opposite unnatural nucleobase analogs unable to form Watson–Crick hydrogen bonds with G. DNA duplexes derived from p53 exon 5 (5′-CCCGGCACCCGC[¹⁵N₃, ¹³C₁-G]TCCGCG-3′) containing cytosine (C), difluorotoluene (Dft), abasic site (abasic), or perimidin-2-one nucleoside (dPER) opposite the labeled G were treated with (±)-anti-BPDE (N = 4) (A) or (−)-anti-BPDE (N = 6) (B), and the extent of N²-BPDE-dG adduct formation at the isotopically tagged guanine was determined by HPLC-ESI⁺-MS/MS. Insert: pie charts showing the relative contributions of (−)-trans-N²-BPDE-dG, (+)-cis-N²-BPDE-dG, (−)-cis-N²- BPDE-dG and (+)-trans-N²-BPDE-dG isomers. * = significantly different (P < 0.05).

Figure 7.

Low temperature (77 K) fluorescence origin band spectra of trans-anti BPT obtained in the absence and in the presence of DNA duplexes containing a central C:G, ^MeC:G or dPER:G base pair. Spectra in (A) were obtained with a λ_ex of 346 nm and a 60 ns delay time of the observation window immediately after mixing BPT with a 5-fold molar excess of DNA. Curves a–c correspond to the fluorescence spectra of BPT mixed with DNA duplexes containing C, ^MeC or dPER, respectively. Spectrum d corresponds to BPT alone. Spectra in (B) were obtained with λ_ex of 355 nm and otherwise identical conditions as in (A). Curves a′–c′ correspond to the spectra of BPT mixed with DNA duplexes containing C, ^MeC, or dPER, respectively. (C) shows the difference between the emission spectra obtained at 346 and 355 nm (a* = a′–a, b* = b′–b, and c* = c′–c).

Figure 8.

Intercalation of the pseudo-diequatorial conformer of (−)-anti-BPDE above (A) and below the plane of the ^MeC:G base pair (B) prior to nucleophilic attack to form a covalent bond with the N² position of guanine. The sequence that was used in the modeling study was an 11-mer representing the experimental sequence used in the paper: 5′-CCCGC[G]TCCGC-3′/3′-GGGC G[^MeC]AGGCG-5′ (where the ^MeC:G base pair is indicated by the bracketed residues). Hydrogen bonded heavy atom donor–acceptor distances are indicated by white dotted lines, and the distances between C10 of BPDE epoxide and the N² exocyclic amine of the reactive guanine (i.e. d1 in Supplementary Table S5) are indicated by a yellow dashed line. Note the formation of a hydrogen bond between the BPDE epoxide oxygen and the exocyclic amino group of the 5′-flanking guanine (A), suggesting that the transition state of the reaction is stabilized by the DNA architecture.