Visualization of focal nuclear sites of DNA repair synthesis induced by bleomycin in human cells

In this study, we examined DNA repair synthesis in human cells treated with the radiomimetic drug bleomycin, which efficiently induces double-strand b reaks (DSBs). Using tyramidebiotin to amplify fluorescent signals, discrete nuclear foci from the incorporation of 5-iododeoxyuridine (IdU) were detec ted in proliferating human cells treated with bleomycin. We believe this co mes from the repair of DSBs. An increase in the number of foci (>5 per nucl eus) was detected in a major fraction (75%) of non-S-phase cells labeled fo r 30 min with IdU I h after the end of bleomycin treatment. The fraction of cells with multiple IdU-containing foci was found to decrease IS h after t reatment. The average number of foci per nucleus detected I h after bleomyc in treatment was found to decrease twofold between I and 3.5 h, indicating that the foci may be associated with the slow component of DSB repair. The presence of DSBs in bleomycin-treated cells was confirmed using antibodies against phosphorylated histone H2AX (gamma -H2AX), which is strictly associ ated with this type of DNA damage. After treatment with bleomycin, non-S-ph ase cells also displayed heterogeneous nuclear foci containing tightly boun d proliferating cell nuclear antigen (PCNA), suggesting an ongoing process of unscheduled DNA synthesis. PCNA is known to be involved in base excision repair, but a fraction of the PCNA foci may also be associated with DNA sy nthesis occurring during the repair of DSBs. (C) 2001 by Radiation Research Society.