| Co-Investigator(Kenkyū-buntansha) |
KUBO Kihei Osaka Prefect. Univ., College of Agriculture, Professor, 農学部, 教授 (40117619)
TERATO Hiroaki Hiroshima Univ., Faculty of Science, Research Associate, 理学部, 助手 (00243543)
OHYAMA Yoshihiko Hiroshima Univ., Faculty of Science, Associate Professor, 理学部, 助教授 (30169081)
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| Research Abstract |
Endonuclease III (Endo III) of Escherichia coli is known to be a DNA repair enzyme with a relatively broad specificity for oxidative pyrimidine lesions. The cDNA of a mouse Endo 111 homologue (mNTH1/mNTHL1) was cloned from a mouse T-cell cDNA library. The cDNA was 1025 nucleotide long and encoded a protein consisting 300 amino acids with a predicted molecular mass of 33.6 kDa. The recombinant mNTH1 protein with a HisィイD26ィエD2 tag was over expressed in a nth nei double mutant of Escherichia coli and purified to apparent homogeneity. The expressed mNTH1 protein released tritium labeled-thymine glycol from DNA, showing an N-glycosylase activity. mNTH1 also recognized thymine glycol, urea residues, and abasic sites specifically introduced into oligonucleotide substrates, generating P-elimination products. Thus, mNTH1 is a bifunctional repair enzyme with N-glycosylase and β-lyase activities.
7, 8-Dihydro-8-oxoguanine (8-oxoG) and 2, 6-diamino-4-hydroxyformamido-pyrimidine (Fapy) are major DNA lesions formed by reactive oxygen species and involved in mutagenic and/or lethal events in cells. Both lesions are repaired by hOGG1 and Fpg in human and Escherichia coli cells, respectively. The repair activities of hOGG1 and Fpg were compared using defined oligonucleotides containing 8-oxoG and a methylated analog of Fapy (me-Fapy) at the same site. The kィイD2catィエD2/KィイD2mィエD2 values of hOGG1 for 8-oxoG and me-Fapy were comparable and this was also the case for Fpg. However the kィイD2catィエD2/KィイD2mィエD2 values of hOGG1 for both lesions were approximately 80-fold lower than those of Fpg. hOGG1 and Fpg showed distinct preferences of the base opposite 8-oxoG, with the activity differences being 19.8 (hOGG1) and 12 (Fpg)-fold between the most and least preferred bases. Such preferences were almost abolished and less than 2-fold for both enzymes when me-Fapy was a substrate, suggesting that, unlike 8-oxoG, me-Fapy is not subjected to paired base-dependent repair.
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