Dependence of DNA–Protein Cross-Linking via Guanine Oxidation upon Local DNA Sequence As Studied by Restriction Endonuclease Inhibition
Abstract
Oxidative damage plays a causative role in many diseases, and DNA-protein cross-linking is one important consequence of such damage. It is known that GG and GGG sites are particularly prone to one-electron oxidation, and here we examined how the local DNA sequence influences the formation of DNA-protein cross-links induced by guanine oxidation. Oxidative DNA-protein cross-linking was induced between DNA and histone protein via the flash quench technique, a photochemical method that selectively oxidizes the guanine base in double-stranded DNA. An assay based on restriction enzyme cleavage was developed to detect the cross-linking in plasmid DNA. Following oxidation of pBR322 DNA by flash quench, several restriction enzymes (PpuMI, BamHI, EcoRI) were then used to probe the plasmid surface for the expected damage at guanine sites. These three endonucleases were strongly inhibited by DNA-protein cross-linking, whereas the AT-recognizing enzyme AseI was unaffected in its cleavage. These experiments also reveal the susceptibility of different guanine sites toward oxidative cross-linking. The percent inhibition observed for the endonucleases, and their pBR322 cleavage sites, decreased in the order: PpuMI (5'-GGGTCCT-3' and 5'-AGGACCC-3') > BamHI (5'-GGATCC-3') > EcoRI (5'-GAATTC-3'), a trend consistent with the observed and predicted tendencies for guanine to undergo one-electron oxidation: 5'-GGG-3' > 5'-GG-3' > 5'-GA-3'. Thus, it appears that in mixed DNA sequences the guanine sites most vulnerable to oxidative cross-linking are those that are easiest to oxidize. These results further indicate that equilibration of the electron hole in the plasmid DNA occurs on a time scale faster than that of cross-linking.
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Independent proofs support covalent attachment of nucleophilic amino acids to guanine radical cation upon one-electron oxidation of oligonucleotides mediated by Type I photosensitizer, flash quench, or biphotonic ionization.
“Other independent proofs that support the covalent attachment of nucleophilic amino acids to Gua •+ upon one-electron oxidation of oligonucleotides mediated by a Type I photosensitizer, the flash quench technique or biphotonic ionization, are available (219-222).”
From Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions<sup>†</sup> (2012) · cites this paper as [221]
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- Authors
- Madison, A. L., Perez, Z. A., To, P., Maisonet, T., Rios, E. V., Trejo, Y., Ochoa-Paniagua, C., Reno, A., & Stemp, E. D. A.
- Affiliations
- Mount St. Mary's University (US)
- Corresponding author
- Stemp, Eric D. A.
- ORCID
- Stemp, Eric D. A.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2011-12-28
- Volume
- 51
- Issue
- 1
- Pages
- 362-369
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2334479606
- Cited by
- 18
- References
- 50
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 2 · 2023: 2 · 2022: 1 · 2021: 1 · 2019: 1 · 2017: 1 · 2014: 3 · 2013: 3 · 2012: 2
- FWCI
- 1.04
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- California Institute of Technology
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Carcinogens and Genotoxicity Assessment
- Keywords
- DNA-protein cross-linking, guanine oxidation, oxidative DNA damage, histones, plasmid DNA, pBR322, restriction sites, one-electron oxidation, G-rich sequences, BamHI, EcoRI
- MeSH terms
- Ascomycota, Base Sequence, Cross-Linking Reagents, DNA Damage, DNA Restriction Enzymes, Guanine, Histones, Hydrolysis, Oxidation-Reduction, Proteins, Oxidative Stress
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