Novobiocin treatment reverses radiation-induced alterations in higher-order DNA structure in L5178Y nucleoids

We have studied the effect of novobiocin treatment on radiation-induced damage and its repair in higher-order DNA structure in two mouse leukemia cell lines differing in their radiosensitivity, L5178Y-R (LY-R) and L5178Y-S (LY-S). We used the fluorescent halo technique to measure alterations in the superhelical density and the topological constraints of DNA in LY-R and LY-S nucleoids. The results for untreated cells show that both cell lines reached maximal DNA unwinding at the same concentration of propidium iodide (PI), whereas LY-S nucleoids were less efficient in their ability to rewind their DNA. The loop size did not differ significantly between the cell lines. Incubation of LY-R and LY-S cells with novobiocin at a concentration which does not influence survival (0.1 mM for 45 min), but inhibits DNA synthesis in LY-R cells (by 28%) to a greater extent than in LY-S cells (by 10%), also causes more DNA unwinding in LY-R nucleoids than in LY-S nucleoids. However, a decreased superhelical density was observed in nucleoids from both cell lines. Novobiocin applied before, and present during, irradiation prevents radiation-induced alterations in DNA supercoiling more efficiently in LY-R than in LY-S cells. The presence of novobiocin during the repair period increased DNA rewinding to levels not significantly different from control values in nucleoids from both cell lines.