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.
Oxidative DNA damage contributes to aging and diseases such as Alzheimer's. Guanine is the most easily oxidized DNA base and therefore is most susceptible to oxidative damage. Bulges in DNA arise from replication and recombination errors and if left unrepaired, cause disease. Here, we are studying the effects of bulges on DNA‐ protein crosslinks. A DNA duplex was assembled using the strand 5’‐TATAGATATGGATATGATAT‐3’, which was fluorescently labeled with Alexafluor 546, and a complementary strand containing a single base bulge, 3’‐ TATACTATACYCTATACTATA‐ 5’, where Y = C, G, A or T. A control strand with no bulge was also synthesized. The GG sequence across from the single DNA base bulge localized the guanine radical at that bulge location. The flash quench technique was used to generate guanine radicals and DNA samples were analyzed by the gel shift assay. DNA bulges containing purines lead to an increase in DNA‐ protein crosslinking when compared to the well‐matched DNA. Thus, structural perturbations such as bulges, when near a guanine base, can influence the amount of oxidative DNA‐protein crosslinking.
Oxidative damage plays a causative role in many diseases and DNA‐protein cross‐linking is one important consequence of such damage. Here we examined how local DNA sequence influences DNA‐protein cross‐link formation induced by guanine oxidation. Oxidative DNA‐protein cross‐linking was induced via the flash quench technique. Following oxidation of pBR322 DNA by flash quench, several restriction enzymes (PpuMI, BamHI, EcoRI) were used to probe for the expected damage at guanine sites. These three endonucleases were strongly inhibited by DNA‐protein cross‐linking, whereas the activity of the AT‐recognizing enzyme AseI was unaffected. The % inhibition observed for the endonucleases decreased in the order: PpuMI (5’‐GGGTCCT ‐3’ and 5’‐AGGACCC‐3’) > BamHI (5’‐GGATCC‐3’) > EcoRI (5’‐GAATTC‐3’), consistent with the observed and predicted tendencies for guanine to undergo 1‐electron oxidation: 5’‐GGG‐3’ > 5’‐GG‐3’ > 5’‐GA‐3’. Thus, in mixed DNA sequences, the guanine sites most vulnerable to oxidative cross‐linking are easiest to oxidize. Research support was provided by the National Science Foundation, the Denault‐Loring Research Fellowship, and Mount St. Mary's College.
Oxidative DNA damage contributes to aging, cancer, and other degenerative diseases. Guanine is particularly vulnerable to oxidation, creating radicals which form irreversible crosslinks to proteins. Here, we investigate how the reactivity of the guanine radical in the oligonucleotide 5′‐ATATGATATGGATATGATAT‐3′ depends on its base pairing partner(s). We synthesized DNA duplexes with cytosine derivatives or mismatches across from guanine. Guanine radicals were created photochemically and the DNA was analyzed by gel shift experiments. In the study of mismatches, the duplexes contained mismatches across from the 5′‐G of the GG doublet. Under both nondenaturing and denaturing conditions, crosslinking decreased in the order of G:G > G:A > G:T = G:C. The enhanced reactivity for the G:G and G:A mismatches is attributed to a lowered oxidation potential near the GG doublet. Cytosine derivatives crosslinked in the order of 5‐bromocytosine > cytosine > 5‐methylcytosine. These results are consistent with the presence of more of the more reactive radical cation with the 5‐bromocytosine, since it is a weaker base than cytosine or 5‐methylcytosine; melting temperature experiments indicated minimal differences between duplexes with the different cytosines. These results show that the reactivity of guanine in oxidative DNA‐protein crosslinking reactions depends on its base pairing partner.
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