Journal Article

Incorporation of dUMP into DNA is a major source of spontaneous DNA damage, while excision of uracil is not required for cytotoxicity of fluoropyrimidines in mouse embryonic fibroblasts

Sonja Andersen, Tina Heine, Ragnhild Sneve, Imbritt König, Hans E. Krokan, Bernd Epe and Hilde Nilsen

in Carcinogenesis

Volume 26, issue 3, pages 547-555
Published in print March 2005 | ISSN: 0143-3334
Published online March 2005 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/bgh347
Incorporation of dUMP into DNA is a major source of spontaneous DNA damage, while excision of uracil is not required for cytotoxicity of fluoropyrimidines in mouse embryonic fibroblasts

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Uracil may arise in DNA as a result of deamination of cytosine or through incorporation of dUMP instead of dTMP during replication. We have studied the steady-state levels of uracil in the DNA of primary cells and mouse embryonic fibroblast (MEF) cell lines from mice deficient in the Ung uracil-DNA glycosylase. The results show that the levels of uracil in the DNA of Ung−/− cells strongly depend on proliferation, indicating that the uracil residues originate predominantly from misincorporation during replication. Treatment with 5-fluoro-2′-deoxyuridine (5-FdUrd) or 5-fluorouracil (5-FU) gives rise to a dose-dependent increase of uracil in Ung−/− MEFs (up to 1.5-fold) but not in wild-type cells. Interestingly, Ung−/− MEFs accumulate AP-sites as well as uracil in response to 5-FdUrd but not to 5-FU. This accumulation of repair intermediates suggests a loss of tightly co-ordinated repair in the absence of Ung, and correlates with stronger inhibition of cell proliferation in response to 5-FdUrd, but not to 5-FU, in Ung−/− MEFs compared with wild-type cells. However, other cytotoxic effects of these fluoropyrimidines are comparable in both wild-type and Ung-deficient cells, demonstrating that excision of uracil from DNA by the Ung uracil-DNA glycosylase is not a prerequisite for obtaining cytotoxicity.

Keywords: ConA, Concanavalin A; 5-FdUrd, 5-fluoro-2′-deoxyuridine; 5-FU, 5-fluorouracil; LPS, lipopolysaccharide; MEF, mouse embryonic fibroblast; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PI, propidium iodide; SMUG1, single-strand selective monofunctional uracil glycosylases; TS, thymidylate synthase; UDG, uracil-DNA glycosylase

Journal Article.  7272 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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