Chemical and Biochemical Stability of Guanine Lysine Crosslinks Formed by Guanine Oxidation
Authors: Esparza Diana , Mincitar Janette , Tran Nancy , Ramos Annalisa , Miller Kelsey , Stemp Eric 
Published: June 2021

Guanine is the most susceptible DNA base that can be oxidized because it holds the lowest oxidation potential value in comparison to the rest of the DNA bases. Due to the formation of radicals in the guanine base, the stability of the damaged DNA is often compensated with the formation of DNA‐protein crosslinks. DNA‐protein crosslinks are adducts between DNA and protein that are not easily repaired as lesions.Since it has been recognized that the understanding of such crosslinks is of high importance due to the health impacts they lead to, there have been techniques developed to further convey the chemical and molecular interactions of this biological macromolecules under stress. The flash‐quench technique was engineered to study the electron transfer in proteins due to reactions of guanine radicals with DNA. To start the process, a photosensitive Intercalator is excited by visible light and transforms into a powerful oxidant by an oxidative quenching reaction; the newly oxidized intercalator removes an electron from the nearest guanine base. Using Ru(phen)2dppz2+ as the photosensitive intercalator and Co(NH3)5Cl2+, as the quencher, we detected the radical in poly(dG‐dC) using transient absorption spectroscopy and showed its UV‐visible spectrum to be similar to the neutral form. Previous work has shown that guanine oxidation can lead to DNA‐protein crosslinking and here we examined whether such crosslinks are stable under physiological conditions. Using Poly(lysine) as a model protein, we examined the stability of the guanine‐lysine crosslinks under several conditions, subjecting the crosslinked samples to i) Room temperature, ii) High temperature, iii)) High temperature with hot alkali, or iv) digestion with Formamidopyramidine (Fpg), a glycosylase that releases damaged guanine bases such as 8‐oxoguanine from DNA, and Proteinase K, a serine protease. Samples containing DNA, Poly(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 crosslinking using either the chloroform extraction assay or a gel shift assay. Crosslinks were stable for days at room temperature, but high temperature was sufficient to cleave some of the crosslinks; however, treatment with hot alkali (Piperidine) cleaved all of the crosslinks. The gel shift assay was used to monitor the ability of Fpg and Proteinase K to cleave DNA‐protein crosslinks. Treatment with Fpg enzyme impacted the mobility of the DNA crosslinked by converting supercoiled to nicked DNA. However, it is not evident that Fpg excises the crosslinked material. Though, digestion with Proteinase K displayed more nicked DNA as irradiation time increases, which is consistent with cleavage of crosslinks by Proteinase K. Thus, since extreme temperatures are necessary to cleave the crosslinks and Fpg is not efficient in cleaving the DNA‐lysine crosslinks, it appears that guanine lysine crosslinks would persist in vivo.Support or Funding InformationNSF (MCB 0345478), Denault‐Loring Research Fellowship, Mount Saint Mary's University, Title III STEM Grant