Enzymatic repair of 5-formyluracil I. Excision of 5-formyluracil site-specifically incorporated into oligonucleotide substrates by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II)

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen r adicals and photosensitized oxidation. We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair wit h guanine. Therefore, fU can exist in DNA as a correctly paired fU:A form o r an incorrectly paired fU:G form. In this work, fU was site-specifically i ncorporated opposite A in oligonucleotide substrates to delineate the cellu lar repair mechanism of fU paired with A. The repair activity for fU was in duced in Escherichia coli upon exposure to N-methyl-N'-nitro-N-nitrosoguani dine, and the induction was dependent on the alkA gene, suggesting that Alk A (3-methyladenine DNA glycosylase II) was responsible for the observed act ivity. Activity assay and determination of kinetic parameters using purifie d AlkA and defined oligonucleotide substrates containing fU, 5-hydroxymethy luracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by A lkA with an efficiency comparable to that of 7mG, a good substrate for AlkA , whereas hU, another major thymine methyl oxidation products, was not a su bstrate. H-1 and C-13 NMR chemical shifts of 5-formyl-2'-deoxyuridine indic ated that the 5-formyl group caused base C-6 and sugar C-1' to be electron deficient, which was shown to result in destabilization of the N-glycosidic bond. These features are common in other good substrates for AlkA and are suggested to play key roles in the differential recognition of fU, hU, and intact thymine. Three mammalian repair enzymes for alkylated and oxidized b ases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying tha t the mammalian repair activity for fU resided on a yet unidentified protei n. In the accompanying paper (Terato, H., Masaoka, A., Robayashi, M., Fukus hima, S., Ohyama, Y., Yoshida, M., and Ide, H., J. Biol. Chem. 274, 25144-2 5150), possible repair mechanisms for fU mispaired with G are reported.