Interstrand DNA-DNA cross-link formation between adenine residues and abasic sites in duplex DNA

Abstract

The loss of a coding nucleobase from the structure of DNA is a common event that generates an abasic (Ap) site (1). Ap sites exist as an equilibrating mixture of a cyclic hemiacetal and a ring-opened aldehyde. Aldehydes are electrophilic functional groups that can form covalent adducts with nucleophilic sites in DNA. Thus, Ap sites present a potentially reactive aldehyde as part of the internal structure of DNA. Here we report evidence that the aldehyde group of Ap sites in duplex DNA can form a covalent adduct with the N(6)-amino group of adenine residues on the opposing strand. The resulting interstrand DNA-DNA cross-link occurs at 5'-ApT/5'-AA sequences in remarkably high yields (15-70%) under physiologically relevant conditions. This naturally occurring DNA-templated reaction has the potential to generate cross-links in the genetic material of living cells.

Scheme 1

Scheme 2

Figure 1

DNA duplexes used in this study. Ap-containing duplexes were generated from the corresponding dU-containing duplexes by treatment with UDG. Locations where cross-links form in substantial yield are indicated with a (red \).

Figure 2

Molecular model illustrating the proximity of the abasic site aldehyde and N⁶-amino group of 2′-deoxyadenosine in a 5′-ApT sequence in B-DNA. The image was constructed using Pymol and is based on pdb entry 3bse.

Figure 3

Interstrand cross-link formation in duplexes B–D. Lanes 1–5, duplex B; lanes 6–9, duplex C; and lanes 10–14, duplex D. Lanes 1 and 10 contain the ³²P-labeled, uracil-containing oligodeoxynucleotides. Lanes 2, 6, and 11 contain the ³²P-labeled UDG-treated (abasic-site-containing) duplexes without incubation. Lanes 3, 7, and 12 contain the ³²P-labeled, UDG-treated (abasic-site-containing) duplexes subjected to piperidine workup (1 M, 95 °C, 25 min) to cleave the Ap site. Lanes 4, 8, and 13 contain the cross-linking reactions involving incubation of the abasic-site-containing duplexes for 120 h in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) at 37 °C. Lanes 5, 9, and 14 contain the methoxyamine-capping reactions involving incubation of the abasic-site-containing duplexes in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) and CH₃ONH₂·HCl (2 mM) at 37 °C. The ³²P-labeled oligodeoxynucleotides were resolved by electrophoresis on a 20% denaturing polyacrylamide gel, and the radioactivity in each band quantitatively measured by phosphorimager analysis. The numbers on the left side of the image represent our structural assignment of the bands and referring to the structure numbers in Schemes 2 and 3.

Scheme 3

Figure 4

Hydroxyl radical footprinting of duplex I to locate the site of cross-link attachment. Duplex I contains the same core sequence as that in duplex B. (A) Lane 1 is a Maxam–Gilbert G-specific cleavage (sequencing) reaction of the labeled oligodeoxynucleotide strand in duplex I. Lane 2 is an A+G specific cleavage (sequencing) reaction of the labeled oligodeoxynucleotide strand in duplex I. Lane 3 is the hydroxyl radical footprinting reaction of the single stranded nonuracil containing oligodeoxynucleotide. Lane 4 is the hydroxyl radical footprinting reaction of the slow-migrating, cross-link band generated by incubation of duplex I HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) at 37 °C. The ³²P-labeled oligodeoxynucleotides were resolved on a 20% denaturing sequencing gel, and the radioactivity in each band quantitatively measured by phosphorimager analysis. (B) Maxam–Gilbert G-specific cleavage (sequencing) reaction of the labeled oligodeoxynucleotide strand in duplex I. (C) A+G specific cleavage (sequencing) reaction of the labeled oligodeoxynucleotide strand in duplex I. (D) Hydroxyl radical footprinting reaction of the slow-migrating, cross-link band.

Figure 5

Nucleobase replacement experiments reveal that the underlined adenine residue in the 5′-GApT/5′-AAC sequence of duplex B is critical for cross-link formation. Replacement of this adenine residue in duplex B with a guanine or thymine residue in duplexes E and F, respectively, abrogates cross-link formation (lanes 13 and 18). Replacement of the crucial adenine residue with 2-aminopurine in duplex G yields only small amounts of a slower-moving band (lane 8). Lanes 1–5, duplex B, lanes 6–9, duplex G, lanes 10–14, duplex E, and lanes 15–19, duplex F. Lanes 1, 10, and 15 contain ³²P-labeled uracil-containing oligodeoxynucleotide duplexes. Lanes 2, 6, 11, and 16 contain the ³²P-labeled UDG-treated (abasic-site-containing) duplexes without incubation. Lanes 3, 7, 12, and 17 contain the abasic-site-containing duplexes subjected to piperidine workup (1 M, 95 °C, 25 min) to cleave the Ap site. Lanes 4, 8, 13, and 18 contain the cross-linking reactions involving incubation of the abasic-site-containing duplexes in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) at 37 °C. Lanes 5, 9, 14, and 19 contain the methoxyamine-capping reactions involving incubation of the abasic-site-containing duplexes incubated in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM), and CH₃ONH₂·HCl (2 mM) at 37 °C. The ³²P-labeled oligodeoxynucleotides were resolved by electrophoresis on a 20% denaturing polyacrylamide gel and the radioactivity in each band quantitatively measured by phosphorimager analysis.

Figure 6

LC-ESI-MS and MS/MS analysis of the mixture generated by digestion of the cross-linked oligodeoxynucleotide generated by incubation of duplex B in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) at 37 °C. (A) selected-ion chromatogram monitoring the neutral loss of 2-deoxyribose from the dA-AP cross-link. (B) MS/MS arising from the fragmentation of the ion at m/z 368. (C) Possible structures for the ions observed at m/z 368, 252, and 136 in these experiments.

Figure 7

Interstrand cross-link formation in duplex I. Lane 1, 5′-³²P-labeled uracil-containing precursor of oligodeoxynucleotide duplex I. Lane 2, 5′-³²P-labeled abasic-site-containing oligodeoxynucleotide duplex I without incubation. Lane 3, 5′-³²P-labeled abasic-site-containing oligodeoxynucleotide duplex I cleaved by treatment with piperidine (1 M, 95 °C, 25 min). Lane 4, Duplex I incubated in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) at 37 °C. Lane 5, Duplex I incubated with CH₃ONH₂ (2 mM) in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) at 37 °C. The ³²P-labeled oligodeoxynucleotides were resolved on a polyacrylamide gel, and the radioactivity in each band quantitatively measured by phosphorimager analysis.