Deoxyribonucleoside Incorporation during DNA Repair of Carcinogen-induced Damage in Human Diploid Fibroblasts

Incorporation of tritium labeled deoxyribonucleosides during DNA repair was investigated in confluent human diploid fibroblasts (WI-38) in conditioned medium containing hydroxyurea. After damage with either of two proximate carcinogens, N acetoxy 2 acetylaminofluorene or 7 bromomethylbenz(a)anthracene, incorporation of radioactivity into DNA increased 8 to 45 fold when any of the four labeled deoxyribonucleosides was used as a precursor. Similar results were obtained when ultraviolet light damaged cells were allowed to repair in the presence of deoxyadenosine ³H. Repair synthesis was also demonstrable when deoxyguanosine ³H was used as precursor and bromodeoxyuridine was used as a density label for replicating DNA. Chromatography of nucleosides prepared by enzymatic digestion of repaired DNA's labeled with one of the four tritiated deoxyribonucleosides revealed that virtually all of the radioactivity was recoverable as the expected labeled product. Digestion studies of repaired DNA's with snake venom phosphodiesterase suggested that incorporation was not due to an artifact such as terminal addition. On the basis of these findings the authors conclude that, during DNA repair synthesis in human diploid cells, all four deoxyribonucleosides are incorporated. These studies are important for assessment of the role of repair in chemical carcinogenesis since most previous studies have utilized pyrimidine precursors for repair, but chemical carcinogens damage primarily purine moieties.