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)

Aya Masaoka, Hiroaki Terato, Mutsumi Kobayashi, Akiko Honsho, Yoshihiko Ohyama, Hiroshi Ide

Research output: Contribution to journalArticle

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Abstract

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair with guanine. Therefore, fU can exist in DNA as a correctly paired fU:A form or an incorrectly paired fU:G form. In this work, fU was site-specifically incorporated opposite A in oligonucleotide substrates to delineate the cellular repair mechanism of fU paired with A. The repair activity for fU was induced in Escherichia coli upon exposure to N-methyl-N'-nitro-N- nitrosoguanidine, and the induction was dependent on the alkA gene, suggesting that AlkA (3-methyladenine DNA glycosylase II) was responsible for the observed activity. Activity assay and determination of kinetic parameters using purified AlkA and defined oligonucleotide substrates containing fU, 5- hydroxymethyluracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by AlkA with an efficiency comparable to that of 7mG, a good substrate for AlkA, whereas hU, another major thymine methyl oxidation products, was not a substrate. 1H and 13C NMR chemical shifts of 5-formyl- 2'-deoxyuridine indicated 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 bases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying that the mammalian repair activity for fU resided on a yet unidentified protein. In the accompanying paper (Terato, H., Masaoka, A., Kobayashi, M., Fukushima, S., Ohyama, Y., Yoshida, M., and Ide, H., J. Biol Chem. 274, 25144-25150), possible repair mechanisms for fU mispaired with G are reported.

Original languageEnglish
Pages (from-to)25136-25143
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number35
DOIs
Publication statusPublished - Aug 27 1999
Externally publishedYes

Fingerprint

Escherichia coli Proteins
Oligonucleotides
Repair
Substrates
Thymine
DNA-3-methyladenine glycosidase II
5-formyluracil
Methylnitronitrosoguanidine
Oxidation
Guanine
Chemical shift
Kinetic parameters
Sugars
Escherichia coli
Assays
Reactive Oxygen Species

ASJC Scopus subject areas

  • Biochemistry

Cite this

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). / Masaoka, Aya; Terato, Hiroaki; Kobayashi, Mutsumi; Honsho, Akiko; Ohyama, Yoshihiko; Ide, Hiroshi.

In: Journal of Biological Chemistry, Vol. 274, No. 35, 27.08.1999, p. 25136-25143.

Research output: Contribution to journalArticle

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AU - Terato, Hiroaki

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AU - Ide, Hiroshi

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N2 - 5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair with guanine. Therefore, fU can exist in DNA as a correctly paired fU:A form or an incorrectly paired fU:G form. In this work, fU was site-specifically incorporated opposite A in oligonucleotide substrates to delineate the cellular repair mechanism of fU paired with A. The repair activity for fU was induced in Escherichia coli upon exposure to N-methyl-N'-nitro-N- nitrosoguanidine, and the induction was dependent on the alkA gene, suggesting that AlkA (3-methyladenine DNA glycosylase II) was responsible for the observed activity. Activity assay and determination of kinetic parameters using purified AlkA and defined oligonucleotide substrates containing fU, 5- hydroxymethyluracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by AlkA with an efficiency comparable to that of 7mG, a good substrate for AlkA, whereas hU, another major thymine methyl oxidation products, was not a substrate. 1H and 13C NMR chemical shifts of 5-formyl- 2'-deoxyuridine indicated 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 bases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying that the mammalian repair activity for fU resided on a yet unidentified protein. In the accompanying paper (Terato, H., Masaoka, A., Kobayashi, M., Fukushima, S., Ohyama, Y., Yoshida, M., and Ide, H., J. Biol Chem. 274, 25144-25150), possible repair mechanisms for fU mispaired with G are reported.

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