The oxidation of the thymine methyl group can generate 5-formyluracil (FoU). 
Template FoU residues are known to miscode, generating base substitution 
mutations. The repair of the FoU lesion is therefore important in minimizing 
mutations induced by DNA oxidation. We have studied the repair of FoU in 
synthetic oligonucleotides when paired with A and G. In E. coli cell extract, 
the repair of FoU is four orders of magnitude lower than the repair of U and is 
similar for both FoU:A and FoU:G base pairs. In HeLa nuclear extract, the repair 
of FoU:A is similarly four orders of magnitude lower than the repair of uracil, 
although the FoU:G lesion is repaired 10 times more efficiently than FoU:A. The 
FoU:G lesion is shown to be repaired by E. coli mismatch uracil DNA glycosylase 
(Mug), thermophile mismatch thymine DNA glycosylase (Tdg), mouse mismatch 
thymine DNA glycosylase (mTDG) and human methyl-CpG-binding thymine DNA 
glycosylase (MBD4), whereas the FoU:A lesion is repaired only by Mug and mTDG. 
The repair of FoU relative to the other pyrimidines examined here in human cell 
extract differs from the substrate preferences of the known glycosylases, 
suggesting that additional, and as yet unidentified glycosylases exist in human 
cells to repair the FoU lesion. Indeed, as observed in HeLa nuclear extract, the 
repair of mispaired FoU derived from misincorporation of dGMP across from 
template FoU could promote rather than minimize mutagenesis. The pathways by 
which this important lesion is repaired in human cells are as yet unexplained, 
and are likely to be complex.