Abstract
5′,8-Cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) are lesions resulting from hydroxyl radical (HO·) attack on the 5′H of the nucleoside sugar moiety and exist in both 5′R and 5′S diastereomeric forms. Increased levels of cdA and cdG are linked to Nucleotide Excision Repair (NER) mechanism deficiency and mutagenesis. Discrepancies in the damage measurements reported over recent years indicated the weakness of the actual protocols, in particular for ensuring the quantitative release of these lesions from the DNA sample and the appropriate method for their analysis. Herein we report the detailed revision leading to a cost-effective and efficient protocol for the DNA damage measurement, consisting of the nuclease benzonase and nuclease P1 enzymatic combination for DNA digestion followed by liquid chromatography isotope dilution tandem mass spectrometry analysis.
Highlights
Hydroxyl radicals (HO·) are known for their reactivity toward DNA leading to nucleobase modifications and strand breaks
A direct comparison of relative Nucleotide Excision Repair (NER) efficiencies of the four cyclopurines has been determined in the identical sequence contexts and in the same HeLa cell extract preparations (Kropachev et al, 2014)
Modified oligonucleotides and dimers containing the cyclopurines were synthesized and used by different research groups for the development of enzymatic protocols aiming at the quantitative liberation of the lesions as nucleoside units, prior to quantification (Romieu et al, 1998, 1999; Jaruga et al, 2004)
Summary
Hydroxyl radicals (HO·) are known for their reactivity toward DNA leading to nucleobase modifications and strand breaks. The attack at H5′ of DNA by HO· is estimated to occur with a 55% probability over all possible sugar positions, and produces the C5′ radical (Aydogan et al, 2002). 5′,8-Cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′deoxyguanosine (cdG) in their 5′R and 5′S diastereomeric forms are tandem-type lesions observed among the DNA modifications and identified in mammalian cellular DNA in vivo (Chatgilialoglu et al, 2011). Nowadays these lesions are considered as robust biomarkers for oxidative stress and might be resistant to repair machinery in cells.
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