NDX-1 protein hydrolyzes 8-oxo-7, 8-dihydrodeoxyguanosine-5′-diphosphate to sanitize oxidized nucleotides and prevent oxidative stress in Caenorhabditis elegans

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.

Human MTH3 (NUDT18) Protein Hydrolyzes Oxidized Forms of Guanosine and Deoxyguanosine Diphosphates

“…In the overall structures, MTH3 is about twice as large as the other human proteins (MTH1, MTH2, and NUDT5), which are slightly larger than E. coli MutT. MTH3 is similar in size to Pcd1 (ORFYLR151c) of Saccharomyces cerevisiae and NDX-4 of Caenorhabditis elegans, which have capacities to cleave 8-oxo-dGTP and 8-oxo-dGDP, respectively (30,31). When the amino acid sequence similarity was determined based on the whole sequence of MTH3, the highest values were obtained with the NDX-1 and Pcd1 proteins (37 and 23%, respectively).…”

“…We also examined the proteins for a possible relationship between their primary structures and their ranges of substrate specificity. The three human proteins MTH1, MTH3, and NUDT5, as well as the S. cerevisiae protein Pcd1, can cleave various types of oxidized purine nucleotides, whereas the E. coli MutT and C. elegans NDX-1 proteins act strictly on 8-oxoGua-containing nucleotides (27,28,30), yet specific sequence motifs for distinguishing these two groups of proteins have not been identified. The substrate recognition and binding may be achieved by a few amino acid residues located near or within the active center, and thus, elucidation of the three-dimensional structures is essential for solving this problem.…”

AP endonuclease EXO-3 deficiency causes developmental delay and abnormal vulval organogenesis, Pvl, through DNA glycosylase-initiated checkpoint activation in Caenorhabditis elegans

“…To investigate other DNA damaging agents that cause an increase in Pvl in the exo-3 mutants, we evaluated whether Pvl formation is enhanced by oxidative DNA damaging agents such as ndx-1 (RNAi) , ndx-2 (RNAi) and methyl viologen (MV). NDX-1 and NDX-2 hydrolyze 8-oxo-dGDP into 8-oxo-dGMP 24 , 28 . Accordingly, ndx-1 (RNAi) and ndx-2 (RNAi) lead to an increase in 8-oxo-dGDP in the nucleotide pool.…”

“…The presence of 8-oxo-dGDP reduces the 8-oxo-dGTPase activity of NDX-4, causing 8-oxo-dGTP to accumulate in the pool 24 . 8-oxo-dGTP is incorporated to DNA during DNA replication, resulting in the accumulation of 8-oxoG in DNA 24 , 28 . MV generates superoxide radicals, which subsequently cause oxidative lesions in DNA 29 .…”

“…12) and clk-2 (C07H6 . 6) complementary DNA (cDNA) clones were amplified by PCR from a cDNA library using the same 2 primer pairs from a previous study 24 , 28 . The amplified PCR products were subcloned into the plasmid L4440 for bacteria-mediated feeding RNAi (RNAi plasmid).…”