NUDT16 is a (deoxy)inosine diphosphatase, and its deficiency induces accumulation of single-strand breaks in nuclear DNA and growth arrest

Abstract

Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest k(cat)/K(m) values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP.

Figure 1.

NUDT16 selectively binds to XTP, ITP and GTP. (A) Experimental scheme depicting the screen for nucleotide-binding proteins. Proteins in extracts prepared from SH-SY5Y cells were pulled down with NTP-immobilized Sepharose beads and subjected to SDS-PAGE, LC-MS/MS analysis and western blot analysis. (B) Amino acid sequence of NUDT16. Peptides detected by LC-MS/MS analysis in the screen are shown in bold (Mascot Ion Score >45). (C) Each sample, pulled down from the extract (219 µg total protein), was subjected to western blot analysis using anti-NUDT16 (lower panel). An arrowhead indicates signals for NUDT16.

Figure 2.

His-NUDT16 hydrolyzes (d)IDP in preference to other nucleotides. (A) Samples from the purification of recombinant His-NUDT16 were subjected to SDS-PAGE and GelCode Blue Staining. Lane 1, supernatant of E. coli extract; lane 2, flow-through fraction from the His-tag purification; lane 3, eluate from His-tag purification; lane 4, fraction recovered by ammonium sulfate precipitation; lane 5, sample after dialysis; lane 6, eluate from cation exchange chromatography; lane 7, fraction recovered from gel filtration chromatography. (B) IDP (200 µM) was incubated with 400 nM His-NUDT16 for 1 h at 37°C. Reaction products were analyzed by HPLC (lower panel), and were compared with substrate IDP incubated without His-NUDT16 (upper panel). HPLC chromatograms of both samples obtained by absorbance at 249 nm are shown. (C) Nucleoside di- or triphosphates (10 or 100 µM) were incubated with 50 nM His-NUDT16 for 1 h at 37°C. The reaction products were analyzed by HPLC. The graph shows the concentration of each nucleoside monophosphate product.

Figure 3.

NUDT16 is mainly localized in nuclei, and especially in nucleoli. (A) Relative mRNA level after transfection of NUDT16 siRNA. Three days after transfection with siRNAs, cells were harvested. The NUDT16 mRNA level relative to that of the control was determined by real time quantitative RT-PCR and is shown as the mean ± SD of triplicate experiments. (B) Cells were transfected with siRNAs and harvested 2, 4, and 7 days after transfection. Cell extracts (10 µg total protein) were subjected to western blot analysis with anti-NUDT16. An arrowhead indicates signals for NUDT16. (C) Intracellular localization of NUDT16. HeLa MR cells were transfected with control siRNA#1 (a–f) or with NUDT16 siRNA#1 (g–i). The cells were subjected to immunofluorescence microscopy with anti-NUDT16 (green, b and h) and anti-nucleolin (red, c and i). Nuclei were stained with DAPI (blue, a and g). Merged signals are shown in panel d (blue and green), e (green and red) and f (blue, green and red). Bars indicate 10 µm.

Figure 4.

Knockdown of NUDT16 suppresses proliferation of HeLa MR cells. (A) Experimental schedule. HeLa MR cells were transfected with siRNAs on Day 0. The transfected cells were then further incubated for 24 h before replating at a density of 1.5 × 10³/cm². After an additional incubation, the cells were subjected to further analysis. (B) Cell proliferation assay. The cells in (A) were analyzed for cell proliferation. The graph shows the mean ± SD of results from three independent siRNA transfections. Two-way repeated measures ANOVA, P < 0.0001. (C) HeLa MR cells treated with NUDT16 siRNA#1 show slightly abnormal progression of the cell cycle. Three days after siRNA transfection with NUDT16 siRNA#1 or with control siRNA#1, cells were subjected to flow cytometry. Left panels indicate representative histograms of DNA contents in isolated nuclei from these cells. Data are mean ± SD of results from three independent siRNA transfections and were analyzed using Student’s t-test.

Figure 5.

Knockdown of NUDT16 in HeLa MR cells increases the number of inosine residues in RNA and the level of single strand breaks in nuclear DNA. HeLa MR cells were independently transfected with siRNAs three times. Three days after transfection, cells were subjected to the following analysis. (A) Quantification of inosine or deoxyinosine by LC-MS/MS. HeLa MR cells were harvested to determine the levels of inosine and deoxyinosine [dI]. The numbers of deoxyinosine residues [dI] per 10⁶ nucleosides in DNA or inosine residues per 10⁶ guanosine [G] in RNA from three independent transfections are shown. Student’s t-test, P = 0.0125 (RNA). (B) Knockdown of NUDT16 induces the accumulation of ssDNA in nuclei of HeLa MR cells. HeLa MR cells transfected with control siRNA#1 (a–c) or with NUDT16 siRNA#1 (d–f) were subjected to immunofluorescence microscopy with anti-ssDNA (green, b and e). Nuclei were stained with DAPI (blue, a and d). Merged signals are shown in c and f (blue and green). Percentages of ssDNA-positive nuclei among DAPI-positive nuclei are shown in the bar graph. Data are mean ± SD of three independent siRNA transfections. Student’s t-test, P = 0.000252. (C) Comet assay under alkaline conditions. Tail moments of at least 15 cells were calculated for each group and box-and-whisker plots are shown for three independent assays. Mann–Whitney U-test, P < 0.05. (D) Chromosomal abnormality. Transfected cells were prepared as in (B). Mitotic cells with chromosomal abnormalities (a) were defined as cells with chromatid breakage (b; solid arrowheads), chromatid gap (c; open arrowheads) and/or premature separation (d; arrows). These cells were counted and percentages of cells with chromosomal abnormalities among thirty mitotic cells are shown in the bar graph. Chromatid breakage, chromatid gap, and premature separation in control cells were 8%, 2% and 0%, respectively, and in NUDT16 knockdown cells were12%, 4% and 1%, respectively. Data are mean ± SD from three independent siRNA transfections.

Figure 6.

Model of biological roles of NUDT16. NUDT16 eliminates (d)IDP and (d)ITP from the nucleotide pools in cooperation with ITPA. NDK; nucleoside diphosphate kinases, RNR; ribonucleotide reductase.