|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥2,600,000 (Direct Cost: ¥2,600,000)
1. We have isolated two genes from Saccharomyces cerevisiae as a functional homologue of E. coli mutTrgene. One is UBP11, known as a member of deubiquitinating enzyme family, and another is OKFSC151c in genome database. N-terminal regions of their predicted amino acid sequences were observed to be〜70% similarity to Nterminus half of MutT including the MutT-box. Both gene products were able to suppress tiie transversion from AT to CG caused by misincorporation of oxidized nucleotide 8-oxodGTP inE. Coli mutT deficient strain. The yeast Δubp11 and ΔSC151c strains showed 10- and 40-fold higher frequency in spontaneous mutation than the wildtype strain, respectively. At least GST-SC151c protein purified indicated pyrophosphatase activity for both 8-oxodGTP and 2-OHdATP. These results suggest that these candidates may important to suppress mutagenesis caused by oxidized nucleotides in Saccharomyces cerevisiae.
2. Mutations in SODs and Fur, a represser for iron uptake systems, stimulates generation of ・OH, and caused hypermutability of GC→TA and AT→CG in Escherichia coli. We compared distributions of these mutations to those in mutMY and mutT strains, in which GC→TA and AT→CG were predominantly induced, respectively. The hotspots and sequence contexts where AT→CG occurred frequently in sodAB fur strain were almost identical to those in mutT strain, whereas those where GC→TA occurred were quite different from those in mutMY strain. These suggested that AT→CG is due to 8-oxodGTP, while GC→TA is produced by some other lesion(s), e.g. 2-OHdATP. The sequence context of 2-OHdATP-induced mutations inE. Coli was similar to that in sodAB fur strain. The expression of cDNA for human MTH1, which can hydrolyze 2-OHdATP, significantly suppressed the GC→TA mutator phenotype of the sodAB fur strain. These results suggested that 2-OHdATP may be produced intracellularly by highly oxidative stress condition, implicating in mutagenesis.