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J. Biol. Chem., Vol. 275, Issue 45, 35471-35477, November 10, 2000

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Identification of Repair Enzymes for 5-Formyluracil in DNA
Nth, Nei, AND MutM PROTEINS OF ESCHERICHIA COLI^*

Qiu-Mei Zhang, Izumi Miyabe, Yukiko Matsumoto, Katsuhito Kino^§, Hiroshi Sugiyama^§, and Shuji Yonei^¶

From the  Laboratory of Radiation Biology, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan and the ^§ Institute for Biomaterials and Bioengineering, Tokyo Medical and Dental University, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan

5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. Although 5-foU has been reported to be removed from DNA by Escherichia coli AlkA protein in vitro, its repair mechanisms are not fully understood. In this study, we used the borohydride trapping assay to detect and characterize repair activities for 5-foU in E. coli extracts with site-specifically designed oligonucleotides containing a 5-foU at defined sites. The trapping assay revealed that there are three kinds of proteins that form covalent complexes with the 5-foU-containing oligonucleotides. Extracts from strains defective in the nth, nei, or mutM gene lacked one of the proteins. All of the trapped complexes were completely lost in extracts from the nth nei mutM triple mutant. The introduction of a plasmid carrying the nth, nei, or mutM gene into the E. coli triple mutant restored the formation of the corresponding protein-DNA complex. Purified Nth, Nei, and MutM proteins were trapped by the 5-foU-containing oligonucleotide to form the complex in the presence of NaBH₄. Furthermore, the purified Nth, Nei, and MutM proteins efficiently cleaved the oligonucleotide at the 5-foU site. In addition, 5-foU was site-specifically incorporated into plasmid pSVK3, and the resulting plasmid was replicated in E. coli. The mutation frequency of the plasmid was significantly increased in the E. coli nth nei mutM alkA mutant, compared with the wild-type and alkA strains. From these results it is concluded that the Nth, Nei, and MutM proteins are involved in the repair pathways for 5-foU that serve to avoid mutations in E. coli.

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