ABSTRACT
8-oxo-dGTP is generated in the nucleotide pool by direct oxidation of dGTP or phosphorylation of 8-oxo-dGDP. It can be incorporated into DNA during replication, which would result in mutagenic consequences. The frequency of spontaneous mutations remains low in cells owing to the action of enzymes degrading such mutagenic substrates. Escherichia coli MutT and human MTH1 hydrolyze 8-oxo-dGTP to 8-oxo-dGMP. Human NUDT5 as well as human MTH1 hydrolyze 8-oxo-dGDP to 8-oxo-dGMP. These enzymes prevent mutations caused by misincorporation of 8-oxo-dGTP into DNA. In this study, we identified a novel MutT homolog (NDX-1) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. NDX-1 did not hydrolyze 8-oxo-dGTP, 2-hydroxy-dATP or 2-hydroxy-dADP. Expression of NDX-1 significantly reduced spontaneous A:T to C:G transversions and mitigated the sensitivity to a superoxide-generating agent, methyl viologen, in an E. coli mutT mutant. In C. elegans, RNAi of ndx-1 did not affect the lifespan of the worm. However, the sensitivity to methyl viologen and menadione bisulfite of the ndx-1-RNAi worms was enhanced compared with that of the control worms. These facts indicate that NDX-1 is involved in sanitization of 8-oxo-dGDP and plays a critical role in defense against oxidative stress in C. elegans.
Subject(s)
Caenorhabditis elegans/metabolism , Guanosine Monophosphate/analogs & derivatives , Nucleotides/metabolism , Oxidative Stress , Amino Acid Sequence , Animals , Caenorhabditis elegans/drug effects , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cloning, Molecular , Enzyme Activation , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Complementation Test , Guanosine Monophosphate/metabolism , Hydrogen Peroxide/pharmacology , Hydrolysis , Microbial Sensitivity Tests , Molecular Sequence Data , Mutation , Paraquat/pharmacology , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , RNA Interference , Sequence Alignment , Vitamin K 3/pharmacologyABSTRACT
The oxidized nucleotide precursors 7, 8-dihydro-8-oxo-dGTP (8-oxo-dGTP) and 1, 2-dihydro-2-oxo-dATP (2-oxo-dATP) are readily incorporated into nascent DNA strands during replication, which would cause base substitution mutations. E. coli MutT and human homologue hMTH1 hydrolyze 8-oxo-dGTP, thereby preventing mutations. In this study, we searched for hMTH1 homologues in the ascidian Ciona intestinalis using the NCBI-BLAST database. Among several candidates, we focused on one open reading frame, designated as CiMutT, because of its high degree of identity (41.7%) and similarity (58.3%) to the overall amino acid sequence of hMTH1, including the Nudix box. CiMutT significantly suppressed the mutator activity of E. coli mutT mutant. Purified CiMutT had a pyrophosphohydrolase activity that hydrolyzed 8-oxo-dGTP to 8-oxo-dGMP and inorganic pyrophosphate. It had a pH optimum of 9.5 and Mg(++) requirement with optimal activity at 5 mM. The activity of CiMutT for 8-oxo-dGTP was comparable to that of hMTH1, while it was 100-fold lower for 2-oxo-dATP than that of hMTH1. These facts indicate that CiMutT is a functional homologue of E. coli MutT. In addition, the enzyme hydrolyzed all four of the unoxidized nucleoside triphosphates, with a preference for dATP. The specific activity for 8-oxo-dGTP was greater than that for unoxidized dATP and dGTP. These results suggest that CiMutT has the potential to prevent mutations by 8-oxo-dGTP in C. intestinalis.