Chemical and structural characterization of interstrand cross-links formed between abasic sites and adenine residues in duplex DNA.
Bottom Line: A synthetic standard was prepared for the putative nucleoside cross-link remnant 6 in which the anomeric carbon of the 2-deoxyribose residue was connected to the exocyclic N(6)-amino group of dA.These findings establish the chemical structure of the dA-Ap cross-link released from duplex DNA and may provide methods for the detection of this lesion in cellular DNA.Both the nucleoside cross-link remnant 6: and the cross-link in duplex DNA were quite stable at pH 7 and 37°C, suggesting that the dA-Ap cross-link could be a persistent lesion with the potential to block the action of various DNA processing enzymes.
Affiliation: Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, MO 65211, USA.Show MeSH
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Mentions: We also monitored the stability of the dA-Ap cross-link embedded within two different DNA duplexes (Figure 7). The 5′-32P-radiolabeled, cross-linked duplexes A and B were prepared as described previously (20), gel purified and then redissolved in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) and incubated at 37°C. At various times, aliquots were removed, frozen and later subjected to analysis by denaturing polyacrylamide gel electrophoresis (Figure 7 and Supplementary Figure S20). The dA-Ap cross-links within duplexes A and B were quite stable, dissociating with half-lives of 66 and 85 h, respectively (Supplementary Figures S20 and S21), to give the Ap-containing oligonucleotide 1 (Scheme 1) and small amounts of the 3′-4-hydroxy-2-pentenal-5-phosphate cleavage product resulting from β-elimination at the Ap site (1,41,42). In a separate experiment, the amount of remaining cross-linked duplexes A and B was monitored after incubation at 22°C over the course of 96 h, in three different buffers with pH values of 5, 7, or 9. At pH 5, 25 and 40% dissociation of cross-linked duplexes A and B, respectively, into the component single strands was observed. No dissociation of the cross-linked duplexes A and B was observed over the course of 96 h at 22°C at pH 7 or 9 (Supplementary Figure S22).
Affiliation: Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, MO 65211, USA.