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.
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.
Andersen
Sonja
S
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK.
Heine
Tina
T
Sneve
Ragnhild
R
König
Imbritt
I
Krokan
Hans E
HE
Epe
Bernd
B
Nilsen
Hilde
H
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics / Q000502:physiology; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D011743:Pyrimidines / Q000494:pharmacology*; D051981:Uracil-DNA Glycosidase
D003856:Deoxyuracil Nucleotides; D011743:Pyrimidines; D004247:DNA; C007267:2'-deoxyuridylic acid; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase