Nuclease P1-mediated fluorescence postlabeling assay of AAF modified DNA model d(TACGTA) and calf-thymus DNA.
Fluorescence postlabeling assay for DNA damage combines enzymatic digestion of modified DNA to nucleoside monophosphates and fluorescence postlabeling. However, to facilitate the quantitative release of bulky adducts that are not readily obtained as mononucleotides, a different mechanism is essential. In order to test the application potential of nuclease P1-mediated fluorescence postlabeling to assay bulky adduct, d(TACGTA) was reacted with N-acetoxy-2-acetylamino-fluorene. The major product, characterized by nmr as AAF adduct of the guanine moiety at the C-8 position, was used as a DNA model. Nuclease P1 digestion of the modified oligomer excised the adduct in two forms (3:1) which were identified as AAF modified d(pGpT) and dpG respectively by cochromotography with authentic markers. Fluorescence postlabeling assay of AAF modified d(TACGTA) detected both forms of the excised adduct. The application of the overall procedure to assay AAF modified calf-thymus DNA demonstrated that the extension of fluorescence postlabeling technique from the mononucleotide to the dinucleotide version expands the scope of the assay.
Sharma
M
M
Department of Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263.
Jain
R
R
D000099:Acetoxyacetylaminofluorene / Q000494:pharmacology; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000187:drug effects; D004249:DNA Damage; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013050:Spectrometry, Fluorescence / Q000379:methods
D009838:Oligodeoxyribonucleotides; D000099:Acetoxyacetylaminofluorene; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases