['5-Fluorouracil (FU) is one of the mainstays of colon cancer chemotherapy. Although developed as an inhibitor of thymidylate synthase, its cytotoxicity has been linked also to its incorporation into RNA. Surprisingly, although FU is incorporated also into DNA, little is known about its metabolism in this nucleic acid.', 'Using extracts of human cells and circular DNA substrates containing a single FU residue either paired with adenine or mispaired with guanine, we studied the enzymology of FU processing.', 'In nicked circular substrates, FU/G mispairs were efficiently repaired by mismatch repair (MMR). In covalently closed circular DNA, which is refractory to MMR, FU/G repair was initiated by either thymine-DNA glycosylase or uracil-DNA glycosylase, whereas FU/A pairs were processed by UNG. Methylated CpG binding domain 4 protein and single-strand selective monofunctional uracil-DNA glycosylase 1 did not detectably contribute to FU removal; however, because these recombinant enzymes process FU/G and FU/A in oligonucleotide substrates, respectively, they too may be involved in FU metabolism in vivo.'] Is 5-Fluorouracil efficiently removed from DNA by the base excision and mismatch repair systems?