Effect of NUDT5 on 8-oxo-dGDP and 8-oxo-dGTP. (A) Reference nucleotides. (B) 8-Oxo-dGDP without treatment. (C) Treatment of 8-oxo-dGDP with NUDT5. (D) 8-Oxo-dGTP without treatment. (E) Treatment of 8-oxo-dGTP with NUDT5. In these experiments, 5 µM 8-oxo-dGDP or 5 µM 8-oxo-dGTP were incubated with a purified preparation of 0.16 µM human amino-terminal His-tagged NUDT5 protein, under the conditions decribed in the Methods section (C,E), or were left untreated (B,D). The treated and untreated forms were then analysed by high-performance liquid chromatography. Arrowheads indicate peaks for 8-oxo-dGMP (single arrowhead), 8-oxo-dGDP (double arrowhead) and 8-oxo-dGTP (triple arrowhead). 8-Oxo-dGTP, 8-oxo-7,8-dihydrodeoxyguanosine triphosphate; 8-oxo-dGDP, 8-oxo-7,8-dihydrodeoxyguanosine diphosphate.

A model for the exclusion of 8-oxoguanine-containing deoxyribonucleotides from the DNA precursor pool in mammalian cells. 8-Oxo-dGTP and 8-oxo-dGDP, which are produced by the oxidation of dGTP and dGDP, respectively, are interconverted by the actions of nucleoside diphosphate kinase (a) and nucleoside triphosphatase (b). 8-Oxo-dGTP is misincorporated into DNA by DNA polymerase (c) to produce mutations. NUDT5 degrades 8-oxo-dGDP to 8-oxo-dGMP, a unusable form for DNA synthesis, and MTH1 cleaves 8-oxo-dGTP to 8-oxo-dGMP. As the activity of MTH1 is inhibited by 8-oxo-dGDP, NUDT5 works in two ways: first, to reduce the amount of substrate for 8-oxo-dGTP synthesis and, second, to promote the cleavage of 8-oxo-dGTP by MTH1. 8-oxo-dGTP, 8-oxo-7,8-dihydrodeoxyguanosine triphosphate; 8-oxo-dGDP, 8-oxo-7,8-dihydrodeoxyguanosine diphosphate.

NUDT5 and MutT-related proteins. (A) Comparison of amino-acid sequences of Escherichia coli MutT and human MTH1 and NUDT5 proteins (Sekiguchi & Tsuzuki, 2002). The 23-residue modules that were previously identified in MutT-related proteins are shown in the lower panel, in which amino-acid residues conserved in all three proteins are indicated by bold letters. Amino acids that could not be replaced by any other amino acid without losing 8-oxo-dGTPase activity are indicated by dots above the columns (Fujii et al., 1999; Shimokawa et al., 2000). (B) SDS–polyacrylamide gel electrophoresis analysis of purified NUDT5 proteins. Lane 1, molecular weight markers. Lane 2, amino-terminal His-tagged NUDT5. Lane 3, carboxy-terminal His-tagged NUDT5. Lane 4, C-terminal His-tagged NUDT5, treated with Factor Xa. Lane 5, an authentic NUDT5 protein, purified on an anti-NUDT5 IgG affinity column. Lane 6, Ni–NTA-agarose bound fraction from M15 cells transfected with vector only (negative control). In lanes 2–5, 0.5 µg of protein was loaded. For lane 6, the same amount of protein from the cell extracts as that used for the purification of 0.5 µg of N-terminal His-tagged NUDT5 was loaded. 8-oxo-dGTP, 8-oxo-7,8-dihydrodeoxyguanosine triphosphate.

Suppression of A:T to C:G transversions by the expression of NUDT5 complementary DNA. (A) Escherichia coli CC101T (mutT⁻) cells containing plasmid pQE30 (vector). (B) E. coli CC101T (mutT⁻) cells containing plasmid PQE30::NUDT5 (cDNA). (C) Mutation rates, as determined by lacZ reversion, in three independent experiments. mutT⁺ and mutT⁻ cells are of E. coli strains CC101 and CC101T, respectively, and carry either pQE30 (vector) or PQE30::NUDT5 (cDNA).