Analysis of radiation-induced DNA double-strand breaks misrepair is not compromized by broken DNA in human fibroblasts.
It has been suggested that the technique for measuring repair fidelity of radiation-induced DNA double-strand breaks (DSBs) using Southern blotting and hybridization to defined regions of the genome could be compromised by broken or poorly-digested DNA. Since misrepair of DNA DSBs is an important aspect of radiation-induced chromosome aberrations, mutations, and cell killing, we checked for such a supposition in non-transformed human fibroblasts. DSB misrepair was assessed in a NotI-cleavable DNA fragment of 3.2 Mbp located on the long arm of chromosome 21 and detected by D21S1 probe. We hypothesized that the suggested DNA degradation, whether spurious in nature or the results of irradiation-induced phenomena such as apoptosis and/or necrosis, should be detectable with or without NotI restriction enzyme treatment. When the DNA embedded in agarose plugs was separated by electrophoresis without prior NotI restriction, no significant difference was observed in the relative amount of migrating DNA between the control (no irradiation) and 24 h of repair following 80 Gy irradiation. Furthermore, only about 10% of the total signal was located below the 3.2 Mbp band. This suggests that the amount of DNA fragmentation due to biological (apoptosis or necrosis) or technical processes was negligible. The Tunel assay supported these results, as there was little to no apoptosis detectable in these fibroblasts up to 24 h after irradiation. We conclude that in primary human fibroblasts, the NotI method for measuring radiation-induced misrepair is not compromised by DNA degradation.
Alsbeih
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Radiation Biology Laboratory, Biomedical Physics Department MBC-03 KFSHRC, P.O. Box 3354, 11211 Riyadh, Kingdom of Saudi Arabia. galsbeih@kfshrc.edu.sa
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D017209:Apoptosis; D004247:DNA / Q000032:analysis* / Q000528:radiation effects*; D004249:DNA Damage; D015342:DNA Probes; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D005347:Fibroblasts / Q000528:radiation effects*; D006801:Humans; D009693:Nucleic Acid Hybridization; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases