Assessing the Formation of Purine Lesions in Mitochondrial DNA of Cockayne Syndrome Cells

Mitochondrial (mt) DNA and nuclear (n) DNA have known structures and roles in cells; however, they are rarely compared under specific conditions such as oxidative or degenerative environments that can create damage to the DNA base moieties. Six purine lesions were ascertained in the mtDNA of wild type (wt) CSA (CS3BE–wtCSA) and wtCSB (CS1AN–wtCSB) cells and defective counterparts CS3BE and CS1AN in comparison with the corresponding total (t) DNA (t = n + mt). In particular, the four 5′,8–cyclopurine (cPu) and the two 8–oxo–purine (8–oxo–Pu) lesions were accurately quantified by LC–MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. The 8–oxo–Pu levels were found to be in the range of 25–50 lesions/107 nucleotides in both the mtDNA and tDNA. The four cPu were undetectable in the mtDNA both in defective cells and in the wt counterparts (CSA and CSB), contrary to their detection in tDNA, indicating a nonappearance of hydroxyl radical (HO•) reactivity within the mtDNA. In order to assess the HO• reactivity towards purine nucleobases in the two genetic materials, we performed γ–radiolysis experiments coupled with the 8–oxo–Pu and cPu quantifications on isolated mtDNA and tDNA from wtCSB cells. In the latter experiments, all six purine lesions were detected in both of the DNA, showing a higher resistance to HO• attack in the case of mtDNA compared with tDNA, likely due to their different DNA helical topology influencing the relative abundance of the lesions.