Identification and characdterization of DNA glycosylases that recognize and remove oxidative base damage in DNA

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(1) Reactive oxygen species cause a wide variety of oxidative modifications to purines and pyrimidines in DNA. Bacteria and eukaryotes have evolved base excision repair mechanisms for oxidative base damage in DNA. E.coli has three kinds of DNA glycosylase, MutM, Nth and Nei, that recognize and remove oxidatively damaged bases from DNA. These DNA glycosylases are able to recognize and remove 5-formyluracil and 5-hydroxymethyluracil in DNA. However, several evidence showed that there are residual activities in crude extract from E.coli mutM nth nei triple mutant. In this study, we identified the abilities of novel enzyme(s) to recognize double-stranded oligonucleotides containing 5-foU. (2) 7,8-dihydro-8-oxoguanine (8-oxoG) is the most important product of oxidative base damage in DNA, and causes G:C to T:A transversions in bacteria and mammalian cells. 8-oxoG is repaired by MutM in E.coli and 8-oxoG-DNA glycosylase (Ogg1) in yeast and mammalian cells. In the present study, we identified … More and characterized an ascidian homolog of the human hOgg1 in Ciona intestinalis (CiOgg1). Introduction of the CiOgg1 gene significantly reduced the frequency of spontaneous G:C to T:A transversions in E.coli mutM mutY strain. Purified GST-CiOgg1 fusion protein had 8-oxoG DNA glycosylase/AP lyase activity. It formed Schiff base intermediates with 8-oxoG-containing duplex oligonucleotides and removed 8-oxoG preferentially from 8-oxoG/C. The CiOgg1 cleaved 8-oxoG-containing duplex DNA via β-elimination reaction. Furthermore, the expression level of CiOgg1 was compared in various tissues in Ciona intestinalis. The highest expression level was observed in testis. (3) In this study, AP endonuclease in Ciona inteslinalis (CiAPE) was purified and characterized. The properties of CiAPE were compared with those of human APE1. The CiAPE protein was about 34 kDa. It efficiently cleaved tetrahydrofuran (THF)-containing duplex oligonucleotides. In addition, the transformation of E.coli, BW9093 Δxth with CiAPE complemented the H2O2 sensitivity. These results demonstrated that CiAPE has AP endonuclease activity to prevent against oxidative stress in Ciona intestinalis. Less