Enzymatic Release of 5-formyluracil by Mammalian Liver Extracts from DNA Irradiated with Ionizing Radiation

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Abstract
To identify a repair enzyme for 5-formyluracil (5-FU) caused by ionizing radiation in DNA, we used a radiolabelled product-release assay for this thymine-damaged substrate. Double-stranded poly(dA-dT)-poly(dA-dT) was radiolabelled by nick translation with [2-14C]-thymidine triphosphate. The DNA was irradiated with X-rays and incubated with cell extract from mouse liver. Radiolabelled products released from the irradiated DNA into an ethanol-soluble fraction were analysed by reversed-phase hplc. Released 5-FU was detected as a free base during reaction with the cell extract. 5-Formyl-2′-deoxyuridine was not detected in the ethanol supernatant. Boiling the extract at 97°C for 15 min completely abolished its ability to release 5-FU. Similar enzymatic activity was observed with rat liver extract. These results demonstrated that mammalian cells have enzymatic activity to release 5-FU from DNA.