Plant and Cell Physiology Advance Access published online on September 5, 2007
Plant and Cell Physiology, doi:10.1093/pcp/pcm112
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AtNUDX1, an 8-oxo-7,8-dihydro-2’-deoxyguanosine 5’-triphosphate pyrophosphohydrolase, is responsible for eliminating oxidized nucleotides in Arabidopsis
1Department of Food and Nutritional Science, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan
2Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan
Corresponding author: Shigeru Shigeoka. Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan, e-mail: shigeoka{at}nara.kindai.ac.jp, Tel: +81-742-43-8083, Fax: +81-742-43-8083
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Abstract |
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Cellular DNA, RNA, and their precursor nucleotides are at high risk of being oxidized by reactive oxygen species. An oxidized base, 8-oxo-7,8-dihydro-2’-(deoxy)guanosine, can pair with both adenine and cytosine, and thus would cause both replicational and translational errors. Previously, we have reported that an Arabidopsis Nudix hydrolase, AtNUDX1, acts to hydrolyse an oxidized deoxyribonucleotide, 8-oxo-7,8-dihydro-2’-deoxyguanosine 5’-triphosphate (8-oxo-dGTP) (Ogawa et al. J. Biol. Chem. 2005, 280: 25277-25283). Here we showed that 8-oxo-dGTP pyrophosphohydrolase activity is not exhibited by any other Arabidopsis Nudix hydrolase. AtNUDX1 acted on an oxidized ribonucleotide, 8-oxo-GTP, with high affinity (Km: 28.1 µM). In a transcriptional mutational analysis using the LacZ reporter gene, the phenotypic suppression of the lacZ amber mutation in a mutT-deficient Escherichia coli strain caused by the misincorporation of 8-oxo-GTP into the mRNA was significantly diminished by expression of AtNUDX1. These findings suggest that AtNUDX1 prevents transcriptional errors in vivo. A confocal microscopic analysis using GFP fusion protein demonstrated that AtNUDX1 is distributed in the cytosol, where exists the main pool of nucleotides in the cells. the base excision repair pathway was located in the chloroplasts. The level of 8-oxo-guanosine in genomic DNA was significantly increased in knockout nudx1 plants compared with wild-type plants under normal and oxidative stress (3 µM paraquat) conditions. The results obtained here indicate that AtNUDX1 functions in cellular defense against oxidative DNA and RNA damage through the sanitization of their precursor pools in the cytosol in Arabidopsis cells.
Keywords: 8-oxo-dGTP - 8-oxo-GTP - sanitization of nucleotide pool - Nudix hydrolase - oxidative stress
These authors contributed equally to this work.
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