Mutations in DNA are caused by oxidative stress and can lead to aging and various cancers. Guanine is susceptible to oxidation because it has the lowest oxidation potential of the DNA bases, and guanine oxidation leads to DNA-protein cross-links. Here, we examined whether such cross-links are stable under various conditions. Poly-L-lysine was used as a model protein to examine the stability of the crosslink at room temperature, high temperature, and high temperature with hot alkali. We also tested the stability of the cross-link to digestion with Fpg, a bifunctional DNA glycosylase that is capable of excising the damaged base and cleaving the phosphodiester bonds. Samples containing DNA, lysine, Ru(phen)2dppz2+ [phen = 1,10-phenanthroline, dppz = dipyridophenazine] and Co(NH3)5Cl2+ were irradiated with the 442 nm output of a HeCd laser to oxidize the DNA by the flash quench method. The samples were then analyzed for cross-linking using both the chloroform extraction assay and the gel shift assay. By the chloroform extraction assay, the cross-link was found to be stable at room temperature. In contrast, incubation at 100°C with piperidine cleaved nearly all the cross-links whereas the 100°C treatment without alkali or digestion with Fpg cleaved only some of the cross-links. Lastly, agarose gel electrophoreses with irradiated samples of pUC19 plasmid showed a band of free DNA that shifted toward the wells upon irradiation. Treatment of irradiated samples with Fpg gave rise to different behavior. At lower irradiation times, a band of slightly lower mobility, probably nicked DNA, is observed, consistent with cleavage of the cross-link. At the highest irradiation time, the DNA runs as a smear, indicating it was heavily fragmented by the enzyme. Thus, since high temperatures and Fpg enzyme are not fully efficient in cleaving the cross-links, it appears that guanine-lysine cross-links could persist in vivo.

Chemical and biochemical stability of guanine lysine cross-links formed by guanine oxidation

249th National Meeting (2015)

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