ENZYMATIC RELEASE OF 5-FORMYLURACIL BY MAMMALIAN LIVER EXTRACTS FROM DNA IRRADIATED WITH IONIZING-RADIATION

To identify a repair enzyme for 5-formyluracil (5-FU) caused by ionizi ng radiation in DNA, we used a radiolabelled product-release assay for this thymine-damaged substrate. Double-stranded poly(dA-dT)-poly(dA-d T) was radiolabelled by nick translation with [2-C-14]-thymidine triph osphate. The DNA was irradiated with X-rays and incubated with cell ex tract from mouse liver. Radiolabelled products released from the irrad iated DNA into an ethanol-soluble fraction were analysed by reversed-p hase hplc. Released 5-FU was detected as a free base during reaction w ith the cell extract. 5-Formyl-2'-deoxyuridine was not detected in the ethanol supernatant. Boiling the extract at 97 degrees C for 15 min c ompletely abolished its ability to release 5-FU. Similar enzymatic act ivity was observed with rat liver extract. These results demonstrated that mammalian cells have enzymatic activity to release 5-FU from DNA.