32P-postlabeling measurement of adenine N-1-oxide in cellular DNA exposed to hydrogen peroxide.
A 32P-postlabeling assay has been developed for monitoring the formation within DNA of adenine N-1-oxide, the specific H2O2-mediated oxidation product under nonradical conditions. This has required the chemical synthesis of both 2'-deoxyadenosine N-1-oxide 3'-monophosphate and 2'-deoxyadenosine N-1-oxide 5'-monophosphate, the substrate and the product of polynucleotide kinase mediated phosphorylation. Isolation of the substrate from the other nucleotides was found to be necessary in order to improve the rate of phosphorylation and to prevent self-radiolysis processes. [32P]-2'-deoxyadenosine N-1-oxide 5'-monophosphate was obtained after successive ion exchange and reverse-phase HPLC and was characterized by a microreaction. The sensitivity of the assay, which is close to 1 modified adenine N-1-oxide/10(6) bases, allowed the determination of this lesion within the DNA of cells exposed to nonlethal levels of H2O2.
Mouret
J F
JF
Département de Recherche Fondamentale, Centre d'Etudes Nucléaires de Grenoble, France.
Odin
F
F
Polverelli
M
M
Cadet
J
J
D000225:Adenine / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004269:DNA, Bacterial / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000302:isolation & purification; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000633:toxicity*; D009711:Nucleotides / Q000138:chemical synthesis; D010084:Oxidation-Reduction; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D004269:DNA, Bacterial; D003838:Deoxyadenine Nucleotides; D009711:Nucleotides; D010761:Phosphorus Radioisotopes; C071516:2'-deoxyadenosine N-1-oxide 3'-monophosphate; C030755:adenine-N-oxide; D000255:Adenosine Triphosphate; D004247:DNA; D006861:Hydrogen Peroxide; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D000225:Adenine