Quantitative Classification of DNA Damages Induced by Submicromolar Cadmium Using Oligonucleotide Chip Coupled with Lesion-Specific Endonuclease Digestion

摘要

Implementation of proper analytical tool for systematic investigation and quantitative determination of different classes of cadmium ion-induced DNA damages, especially at low metal ion concentrations, is still lacking. Using lesion-specific enzymes that cleave DNA at specific classes of damage and a fluorometric approach developed for quantifying fluorophore-labeled oligonudeotides bound to chip surfaces, we determined the frequencies of different lesions (strand breaks, oxidized purines, oxidized pyrimidines, or abasic sites) induced by submicromolar Cd2+. Cd2+-treated oligonucleotide chips were digested with various endonucleases (Fpg protein, endonuclease III, endoudease IV), producing a de novo single strand break (SSB) at their substrate modifications. The frequency of SSB and double strand break (DSB) was computed from the difference of pre- and post-Cd2+-treatment oligonucleotide coverage on the chip. While the frequency of SSBs and oxidized bases were successfully quantified even at 0.5 [M of Cd2+, DSB frequency could be easily quantitated at 8.7 fltA [Cd2+]. The numbers of abasic sites were below the oligonucleotide detection limit (2.4 amole; equivalent to 0.24 fM for a reaction volume of 100 /lL). SSBs were found to constitute about 85-90% of single strand damages, while oxidized bases comprise only 4-7% of the total at 0.9 to 8.7 flM [Cd2+].