Ntg1 and Ntg2 proteins as 5-formyluracil-DNA glycosylases/AP lyases in Saccharomyces cerevisiae.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Zhang
Q M
QM
Laboratory of Radiation Biology, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
Hashiguchi
K
K
Kino
K
K
Sugiyama
H
H
Yonei
S
S
D001483:Base Sequence; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D005800:Genes, Fungal; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014498:Uracil / Q000031:analogs & derivatives* / Q000378:metabolism
D004271:DNA, Fungal; D017382:Reactive Oxygen Species; D029701:Saccharomyces cerevisiae Proteins; C007777:5-formyluracil; D014498:Uracil; D009699:N-Glycosyl Hydrolases; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C101825:NTG1 protein, S cerevisiae; C116827:NTG2 protein, S cerevisiae