Enzymatic release of 5-formyluracil by mammalian liver extracts from DNA irradiated with ionizing radiation

• Published in: International journal of radiation biology Vol. 68; no. 6; p. 603
• Main Authors: Zhang, Q M, Fujimoto, J, Yonei, S
• Format: Journal Article
• Language: English
• Published: England 1995
• Subjects: Animals; DNA - metabolism; DNA - radiation effects; DNA Repair; Female; Liver - metabolism; Mice; Mice, Inbred ICR; Pentoxyl - analogs & derivatives; Pentoxyl - metabolism; Rats; Tissue Extracts - metabolism; Uracil - analogs & derivatives; Uracil - metabolism
• ISSN: 0955-3002
• DOI: 10.1080/09553009514551601

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 degrees 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.