title: Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: application to gamma-radiation and peroxynitrite.
abstract: We report a highly sensitive method to quantify abasic sites and deoxyribose oxidation products arising in damaged DNA. The method exploits the reaction of aldehyde- and ketone-containing deoxyribose oxidation products and abasic sites with [(14)C]methoxyamine to form stable oxime derivatives, as originally described by Talpaert-Borle and Liuzzi [Reaction of apurinic/apyrimidinic sites with [(14)C]methoxyamine. A method for the quantitative assay of AP sites in DNA, Biochim. Biophys. Acta 740 (1983) 410-416]. The sensitivity of the method was dramatically improved by the application of accelerator mass spectrometry to quantify the (14)C, with a limit of detection of 1 lesion in 10(6) nucleotides in 1 microg of DNA. The method was validated using DNA containing a defined quantity of abasic sites, with a >0.95 correlation between the quantities of abasic sites and those of methoxyamine labels. The original applications of this and similar oxyamine derivatization methods have assumed that abasic sites are the only aldehyde-containing DNA damage products. However, deoxyribose oxidation produces strand breaks and abasic sites containing a variety of degradation products with aldehyde and ketone moieties. To assess the utility of methoxyamine labeling for quantifying strand breaks and abasic sites, the method was applied to plasmid DNA treated with gamma-radiation and peroxynitrite. For gamma-radiation, there was a 0.99 correlation between the quantity of methoxyamine labels and the quantity of strand breaks and abasic sites determined by a plasmid nicking assay; the abasic sites comprised less than 10% of the radiation-induced DNA damage. Studies with peroxynitrite demonstrate that the method, in conjunction with DNA repair enzymes that remove damaged bases to produce aldehydic sugar residues or abasic sites, is also applicable to quantifying nucleobase lesions in addition to strand break products. Compared to other abasic site quantification techniques, the modified method offers the advantage of providing a straightforward and direct measurement of aldehyde- and ketone-containing strand breaks and abasic sites, with the potential for direct labeling in cells prior to DNA isolation.
authors.affiliation: Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Xinfeng
authors.initials: X
authors.lastname: Zhou
authors.forename: Rosa G
authors.initials: RG
authors.lastname: Liberman
authors.forename: Paul L
authors.initials: PL
authors.lastname: Skipper
authors.forename: Yelena
authors.initials: Y
authors.lastname: Margolin
authors.forename: Steven R
authors.initials: SR
authors.lastname: Tannenbaum
authors.forename: Peter C
authors.initials: PC
authors.lastname: Dedon
mesh_terms: D004247:DNA / Q000737:chemistry*; D004249:DNA Damage / Q000528:radiation effects; D005720:Gamma Rays; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010091:Oximes / Q000032:analysis* / Q000737:chemistry; D030421:Peroxynitrous Acid / Q000737:chemistry*
chemical_list: D010091:Oximes; D030421:Peroxynitrous Acid; D004247:DNA