Zhang Q M, Hashiguchi K, Kino K, Sugiyama H, Yonei S
Laboratory of Radiation Biology, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
Int J Radiat Biol. 2003 May;79(5):341-9. doi: 10.1080/0955300032000093119.
5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. The present authors reported previously that MutM, Nth and Nei in Escherichia coli removed 5-foU from DNA. The present study identified 5-foU DNA glycosylases in Saccharomyces cerevisiae in order to clarify the repair mechanisms of 5-foU in eukaryotic cells.
The borohydride-trapping assay and DNA-nicking assay were carried out to detect and characterize the repair activities for 5-foU in extracts from S. cerevisiae with oligonucleotides containing 5-foU at specific sites.
Two proteins in crude extracts from S. cerevisiae formed covalent complexes with oligonucleotides containing site-specific 5-foU in the presence of NaBH4. Extracts from S. cerevisiae strains defective in either the NTG1 or the NTG2 gene lacked either one or the other of these two proteins. Purified Ntg1 and Ntg2 were trapped in such complexes by the 5-foU-containing oligonucleotides in the presence of NaBH4. Furthermore, purified Ntg1 and Ntg2 efficiently cleaved the oligonucleotide at the 5-foU site.
The results indicate that both Ntg1 and Ntg2 are involved in the repair of 5-foU in DNA, and thereby serve to reduce mutations in S. cerevisiae.
5-甲酰尿嘧啶（5-foU）是电离辐射和各种化学氧化剂在DNA中产生的胸腺嘧啶潜在诱变损伤。本作者之前报道过，大肠杆菌中的MutM、Nth和Nei可从DNA中去除5-foU。本研究鉴定了酿酒酵母中的5-foU DNA糖基化酶，以阐明真核细胞中5-foU的修复机制。
采用硼氢化钠捕获试验和DNA切口试验，以检测和表征酿酒酵母提取物对特定位点含有5-foU的寡核苷酸的5-foU修复活性。
在硼氢化钠存在下，酿酒酵母粗提物中的两种蛋白质与含有位点特异性5-foU的寡核苷酸形成共价复合物。NTG1或NTG2基因缺陷的酿酒酵母菌株提取物缺乏这两种蛋白质中的一种或另一种。在硼氢化钠存在下，纯化的Ntg1和Ntg2被含5-foU的寡核苷酸捕获在这种复合物中。此外，纯化的Ntg1和Ntg2在5-foU位点有效地切割寡核苷酸。
结果表明，Ntg1和Ntg2都参与了DNA中5-foU的修复，从而有助于减少酿酒酵母中的突变。
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