Chemical and biochemical stability of guanine lysine cross-links formed by guanine oxidation
Authors:Diana Esparza, Kelsey Miller, Janette Mincitar, Annalisa Ramos
Mentor:Eric Stemp , Department Chair, Physical Sciences, Mount St. Mary's College
Oxidative damage to DNA plays a role in cancer and aging, and guanine is the most easily oxidized base. Previous work has shown that guanine oxidation can lead to DNA-protein cross-linking and here we examined whether such cross-links are stable under physiological conditions. Using poly(lysine) as a model protein, we examined the stability of the guanine-lysine cross-link under several conditions, subjecting the cross-link samples to i) room temperature, ii) high temperature, iii))high temperature with hot alkali, or iv) digestion with Fpg, a glycosylase that releases damaged guanine bases such as 8-oxoguanine from DNA. Samples containing DNA, lysine, Ru(phen)2dppz2+ [phen = phenanthroline, dppz = dipyridophenazine], and Co(NH3)5Cl2+ were irradiated with the 442 nm output of a HeCd laser. The samples, once the crosslinks were induced by flash quench as above, were then analyzed for cross-linking using either the chloroform extraction assay or a gel shift assay. Cross-links were stable for days at room temperature, but high temperature was sufficient to cleave some of the cross-links; however, treatment with hot alkali (piperidine) cleaved all of the cross-links. Lastly, treatment with Fpg enzyme had a modest effect, also cleaving some of the cross-links if the lysine is small. Thus, since extreme temperatures are necessary to cleave the cross-links and the enzyme is not efficient in cleaving the cross-links, it appears that guanine lysine cross-links would persist in vivo.