Chemical structure and properties of interstrand cross-links formed by reaction of guanine residues with abasic sites in duplex DNA

Abstract

A new type of interstrand cross-link resulting from the reaction of a DNA abasic site with a guanine residue on the opposing strand of the double helix was recently identified, but the chemical connectivity of the cross-link was not rigorously established. The work described here was designed to characterize the chemical structure and properties of dG-AP cross-links generated in duplex DNA. The approach involved characterization of the nucleoside cross-link "remnant" released by enzymatic digestion of DNA duplexes containing the dG-AP cross-link. We first carried out a chemical synthesis and complete spectroscopic structure determination of the putative cross-link remnant 9b composed of a 2-deoxyribose adduct attached to the exocyclic N(2)-amino group of dG. A reduced analogue of the cross-link remnant was also prepared (11b). Liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis revealed that the retention times and mass spectral properties of synthetic standards 9b and 11b matched those of the authentic cross-link remnants released by enzymatic digestion of duplexes containing the native and reduced dG-AP cross-link, respectively. These results establish the chemical connectivity of the dG-AP cross-link released from duplex DNA and provide a foundation for detection of this lesion in biological samples. The dG-AP cross-link in duplex DNA was remarkably stable, decomposing with a half-life of 22 days at pH 7 and 23 °C. The intrinsic chemical stability of the dG-AP cross-link suggests that this lesion in duplex DNA may have the power to block DNA-processing enzymes involved in transcription and replication.

Figure 1

Selected regions of ¹H–¹H COSY and ¹H–¹³C HMBC spectra of 14 in CDCl₃ acquired at 500 (¹H) and 126 (¹³C) MHz. Attachment of the 3,5-bis[O-(tert-butyldimethylsilyl)]-2-deoxyribofuranose moiety to N² of the protected dG was established by (a) homonuclear (COSY) coupling of H1″ to N²-H as well as three-bond HMBC coupling of (b) N²-H to C2″ and (c) H1″ to C2.

Figure 2

¹H–¹H NOESY spectrum of 14 in CDCl₃ acquired at 500 MHz with a mixing time of 800 ms; negative peaks are denoted by red curves. The expanded frame shows the strong through-space correlation between H4″ and N²-H of the major α-furanose isomer. Also shown is a cross-peak between H4″ and H1″ of the minor β-furanose isomer; no correlation between H4″ and N²-H of the minor isomer is evident.

Figure 3

Selected regions of ¹H–¹³C HMBC and ¹H–¹H COSY spectra of 9b obtained at 500 (¹H) and 126 (¹³C) MHz, respectively. Correlations between (a) H1″ and C2 in D₂O as well as (b) H1″ and N²-H in DMSO-d₆ indicate that the 2-deoxyribose moiety remained attached to N² of dG following deprotection.

Figure 4

Stability of dG–AP cross-linkage. (A) Synthetic cross-link remnant 9b was incubated at 23 °C in HEPES buffer (pH 7, 50 mM) containing NaCl (100 mM). Disappearance of 9b and concomitant appearance of dG were monitored by HPLC. (B) Gel electrophoretic analysis of purified, unreduced cross-link in duplex A, incubated at 37 °C in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM). The fast-moving fragment at the bottom of the gel corresponds to the 3′-4-hydroxy-2-pentenal-5-phosphate cleavage product resulting from β-elimination at the AP site.

Figure 5

LC-MS/MS and MS/MS/MS for characterization of the dG–AP cross-link remnant that was obtained synthetically (a and c) or released from purified AP-derived cross-link-containing duplex DNA sequence A (b and d). (a, b) Selected-ion chromatograms for monitoring the loss of 2-deoxyribose (i.e., m/z 384 → 268 transition) from [M + H]⁺ ion of the unreduced dG–AP cross-link remnant that was (a) obtained synthetically or (b) liberated from the four-enzyme digestion of purified cross-link-containing duplex DNA. (c, d) Corresponding selected-ion chromatograms for monitoring the formation of [M + H]⁺ ion of guanine (i.e., m/z 384 → 268 → 152 transition) from the ion of m/z 268 observed in MS/MS. Depicted in the insets are the corresponding MS/MS and MS/MS/MS averaged from the retention times of (a, c) 23.7 min and (b, d) 23.0 min. The MS/MS/MS averaged from the two other peaks are displayed in Figure S26. The ion of m/z 250 observed in panel d is attributed to isobaric interference.

Figure 6

LC-MS/MS and MS/MS/MS for characterization of reduced dG–AP cross-link remnant that was obtained synthetically or released from NaCNBH₃-treated cross-linked duplex DNA. Shown are selected-ion chromatograms for monitoring the loss of a 2-deoxyribose (i.e., m/z 386 → 270 transition) from the [M + H]⁺ ion of reduced dG–AP cross-link remnant that was (a) obtained synthetically or (b) released from the 4-enzyme digestion of reduced product of the purified cross-linked duplex A. (c, d) Corresponding selected-ion chromatograms for monitoring the further loss of H₂O from the ion at m/z 270 observed in MS/MS (i.e., m/z 386 → 270 → 252 transition). (Insets) Corresponding MS/MS and MS/MS/MS averaged from retention times of (a, c) 20.5 and (b, d) 20.8 min.

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