Stable Interstrand Cross-Links Generated from the Repair of 1, N6-Ethenoadenine in DNA by α-Ketoglutarate/Fe(II)-Dependent Dioxygenase ALKBH2

Abstract

DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N⁶-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N⁶-amino group of adenine or the N⁴-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.

Figure 1.

Proposed general reaction mechanism of ALKBH2 dealkylating εA, as well as ICL structures. DNA₁: εA lesion containing strand. DNA₂: complementary strand.

Figure 2.

High-resolution ESI-TOF MS spectra, HPLC chromatograms, and denaturing polyacrylamide gel electrophoresis of starting materials and products. The observed m/z values represent the oligonucleotides under their −2 to −4 charge states. (a–d) Peak envelope of oligonucleotide species in MS and (e–h) retention times of the oligonucleotide species obtained from the extraction of the peak envelope of a given oligonucleotide. (a,e) Starting material containing εA: 10mer 5′-GACCTεATGCC-3′ (5′-TεAT-3′), (b,f) fully repaired nucleotide: 10mer 5′-GACCTATGCC-3′, and (c,g) complementary strand: 20mer 3′-AATATCTGGAAACGGTTTAA-5′; and (d,h) ICL generated in the reaction. (i) Denaturing polyacrylamide gel electrophoresis of the ICL adduct generated from εA in duplex after repair by ALKBH2. Lanes 1 and 5: 20mer, 40mer, and 60mer oligonucleotides as DNA ladders; lane 2: 5′-AAA-3′ 20mer complementary strand; lane 3: duplex (5′-TXT-3′/3′-AAA-5′, X = εA) was incubated under standard ALKBH2 reaction conditions; lane 4: duplex (5′-TXT-3′/3′-AAA-5′) was incubated using the Co(II) ion instead of Fe(II) ion (no-reaction control); lane 6: 5′-TTT-3′ 20mer complementary strand; lane 7: duplex (5′-AXA-3′/3′-TTT-5′) was incubated under standard reaction conditions; and lane 8: duplex (5′-AXA-3′/3′-TTT-5′) was incubated using the Co(II) ion instead of the Fe(II) ion (noreaction control).

Figure 3.

Computed frequencies of reactive conformations suitable for interstrand (left) or intrastrand (right) cross-link formation. Sequence context: 5′-NXT-3′/3′-NTA-5′ (A,B); 5′-NXG-3′/3′-NTC-3′ (C,D); 5′-NXC-3′/3′-NTG-5′ (E,F); or 5′-NXA-3′/3′-NTT-5′ (G,H). Reactive conformations involving C10 (blue) or C11 (orange) of the epoxide are provided, as well as the sum for both sites (purple).

Figure 4.

MD representative reactive conformations for the predicted dominant ICLs between the epoxide intermediate (X) and the amino group of interstrand A (top) and C (bottom) nucleobases. Average electrophile−nucleophile distance (r(N_nuclC_elec), A) and angle of attack (∠(N_nuclC_elecO_leave), deg) across all replicas are provided. Top left: N⁶ in A to C10 in epoxide (see Figure 1 for the positions of C10 and C11); top right: N⁶ in A to C11 in epoxide; bottom left: N⁴ in C to C10 in epoxide; and bottom right: N⁴ in C to C11 in epoxide.

Figure 5.

LC-ESI-MS/MS profile of εA epoxide-induced A cross-link (εA-A-CL) and C cross-link (εA-C-CL) in duplexes 5′-TXT-3′/3′-AAA-5′ (upper panel, a,c,e) and 5′-TXT-3′/5′-ACA-3′ (lower panel, b,d,f). (a,b) Sequence structure of duplexes and chemical structure of εA-A-CL and εA-C-CL; (c,d) integrated chromatograms for εA-A-CL and εA-C-CL for their precursor ions and major product ions. (e,f) MS2 spectrum for εA-A-CL and εA-C-CL with annotations of tentative structures.

Figure 6.

Detection of epoxide-induced A cross-link (εA-A-CL) in HeLa cells exposed to CAA by LC-ESI-MS/MS analysis. (a) Schematic illustration for cell treatment, DNA extraction, and subsequent analysis for εA-A-CL and (b) integrated chromatograms (major and qualifying product ions) for the εA-A-CL standard (upper panel) purified from the duplex 5′-TXT/5′-AAA and εA-A-CL in CAA-exposed cells (lower panel).