Plant and Cell Physiology Advance Access originally published online on September 5, 2007
Plant and Cell Physiology 2007 48(10):1438-1449; 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: E-mail, shigeoka{at}nara.kindai.ac.jp; 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 hydrolyze an oxidized deoxyribonucleotide, 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP). 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 a green fluorescent protein (GFP) fusion protein demonstrated that AtNUDX1 is distributed in the cytosol, where the main pool of nucleotides in the cells exists. 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 - Nudix hydrolase - Oxidative stress - Sanitization of nucleotide pool
Abbreviations: ApnA, diadenosine polyphosphate; DAPI, 4',6-diamidino-2-phenylindole; ECD, electrochemical detector; GFP, green fluorescent protein; KO-nudx1, knockout AtNUDX1 mutants; MS, Murashige and Skoog; MTH, MutT homolog; NUDX, Nudix hydrolases; 8-oxo-G, 8-oxo-7,8-dihydro-2'-guanosine; 8-oxo-(d)GTP, 8-oxo-7,8-dihydro-2'-(deoxy)GTP; PQ, paraquat; ROS, reactive oxygen species; RT–PCR, reverse transcription–PCR; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside.
3These authors contributed equally to this work.
(Received May 19, 2007; Accepted August 21, 2007)
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