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Chemical and structural characterization of interstrand cross-links formed between abasic sites and adenine residues in duplex DNA.

Price NE, Catalano MJ, Liu S, Wang Y, Gates KS - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, MO 65211, USA.

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Above: cross-linked oligonucleotide duplexes used for enzymatic digestion and LC-MS/MS analysis and stability studies (duplexes used in mass spectrometric studies did not contain the 32P label). Location of the dA-Ap cross-links is indicated with the line (\). Below: dissociation of the purified cross-link in duplex A incubated in 50 mM HEPES (pH 7.0) and 100 mM NaCl at 37°C for 0, 0.25, 1, 2, 5, 10, 15 and 21 days (lanes 1–8). Following incubation for the specified time, DNA in the samples was ethanol precipitated, washed and stored at –20°C until analysis by 20% denaturing gel electrophoresis. The amount of remaining cross-link at each time point was quantitatively measured by phosphorimager analysis.
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Figure 7: Above: cross-linked oligonucleotide duplexes used for enzymatic digestion and LC-MS/MS analysis and stability studies (duplexes used in mass spectrometric studies did not contain the 32P label). Location of the dA-Ap cross-links is indicated with the line (\). Below: dissociation of the purified cross-link in duplex A incubated in 50 mM HEPES (pH 7.0) and 100 mM NaCl at 37°C for 0, 0.25, 1, 2, 5, 10, 15 and 21 days (lanes 1–8). Following incubation for the specified time, DNA in the samples was ethanol precipitated, washed and stored at –20°C until analysis by 20% denaturing gel electrophoresis. The amount of remaining cross-link at each time point was quantitatively measured by phosphorimager analysis.

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).


Chemical and structural characterization of interstrand cross-links formed between abasic sites and adenine residues in duplex DNA.

Price NE, Catalano MJ, Liu S, Wang Y, Gates KS - Nucleic Acids Res. (2015)

Above: cross-linked oligonucleotide duplexes used for enzymatic digestion and LC-MS/MS analysis and stability studies (duplexes used in mass spectrometric studies did not contain the 32P label). Location of the dA-Ap cross-links is indicated with the line (\). Below: dissociation of the purified cross-link in duplex A incubated in 50 mM HEPES (pH 7.0) and 100 mM NaCl at 37°C for 0, 0.25, 1, 2, 5, 10, 15 and 21 days (lanes 1–8). Following incubation for the specified time, DNA in the samples was ethanol precipitated, washed and stored at –20°C until analysis by 20% denaturing gel electrophoresis. The amount of remaining cross-link at each time point was quantitatively measured by phosphorimager analysis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4402519&req=5

Figure 7: Above: cross-linked oligonucleotide duplexes used for enzymatic digestion and LC-MS/MS analysis and stability studies (duplexes used in mass spectrometric studies did not contain the 32P label). Location of the dA-Ap cross-links is indicated with the line (\). Below: dissociation of the purified cross-link in duplex A incubated in 50 mM HEPES (pH 7.0) and 100 mM NaCl at 37°C for 0, 0.25, 1, 2, 5, 10, 15 and 21 days (lanes 1–8). Following incubation for the specified time, DNA in the samples was ethanol precipitated, washed and stored at –20°C until analysis by 20% denaturing gel electrophoresis. The amount of remaining cross-link at each time point was quantitatively measured by phosphorimager analysis.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, MO 65211, USA.

Show MeSH
Related in: MedlinePlus