Estimation of DNA damage and repair in tissues of gamma-irradiated animals using the polymerase chain reaction.
Damage and repair of DNA isolated from brain and spleen of gamma-irradiated rats were assayed using the polymerase chain reaction (PCR) method. Damage produced by gamma-radiation in DNA in cells of these tissues of exposed animals was shown to block PCR with the Tth polymerase. This blockage was noted as a decrease in the level of amplification of the fragments of a transcribed gene (beta-actin), an inducible gene (p53), and a nontranscribed one (IgE, heavy chain). The most pronounced decrease in the amplification of the gene fragments was observed on the DNA template isolated from rats immediately after their gamma-irradiation. When DNA was isolated 0.5-5.0 h after exposure, the amplification level was restored, no matter what transcription activity the genes possessed. For comparison, we used in PCR in vitro gamma-irradiated DNA as well as DNA templates with UV-damage, 8-oxy-2;-deoxyguanosine (8-O-dG), and apurinic sites (AP-sites). We found that gamma- and UV-irradiated DNA as well as DNA with AP-sites blocked the Tth polymerase in PCR, whereas 8-O-dG did not effect the level of PCR amplification of gene fragments. The observed changes in the level of PCR amplification of genes on the DNA template from tissues of gamma-irradiated animals are due to various radiation-induced lesions capable of blocking the Tth polymerase. The results show that the PCR method can be used for assaying the integral DNA damage and repair in cells from irradiated animals.
Ploskonosova
I I
II
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292, Russia.
Baranov
V I
VI
Gaziev
A I
AI
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D017931:DNA Primers; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase; D005720:Gamma Rays; D008297:Male; D019384:Nucleic Acid Synthesis Inhibitors; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar
D017931:DNA Primers; D019384:Nucleic Acid Synthesis Inhibitors; D004247:DNA; C068508:Tth polymerase; D004259:DNA-Directed DNA Polymerase