Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells

www.nature.com/scientificreports OPEN received: 16 December 2015 accepted: 16 August 2016 Published: 13 September 2016 Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells Yasuto Yoneshima1,2, Nona Abolhassani1, Teruaki Iyama1, Kunihiko Sakumi1,3, Naoko Shiomi4, Masahiko Mori4, Tadahiro Shiomi4, Tetsuo Noda5, Daisuke Tsuchimoto1,3 & Yusaku Nakabeppu1,3 Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/ protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity. For all organisms, maintenance of the integrity of genomic DNA and its precise transmission from cell to cell and from parents to offspring is fundamental to life. DNA, however, is susceptible to damage from various reactive molecules. Some DNA damage induces cell death or genetic mutation, and causes various disorders in humans, such as aging, cancer and hereditary diseases1,2. Base moieties of nucleic acids, which define genetic information, also suffer various chemical modifications, such as oxidation, deamination, methylation or halogenation3–6 that result in the generation of abnormal bases. These modifications can occur because of endogenous factors, such as reactive oxygen or nitrogen species, or after exposure to exogenous factors, such as ionizing radiation, ultraviolet light or chemical agents3–6. Various enzymatic reactions also generate abnormal bases in nucleic acids7,8. Direct modification of normal bases already incorporated in DNA is one of two main pathways for the accumulation of abnormal bases in DNA. The second pathway is the incorporation of abnormal deoxynucleoside triphosphates from the nucleotide pool into newly synthesized DNA during its replication. To avoid deleterious effects of the abnormal nucleotides, cells are equipped with specific enzymes to hydrolyse the abnormal nucleoside triphosphates to the corresponding monophosphates. These enzymes are known as nucleotide pool sanitizing enzymes9–11. Deoxyinosine (dI) is an abnormal nucleoside and has hypoxanthine as its base moiety. Hypoxanthine is generated by oxidative deamination of adenine, which occurs in the presence of nitrous acid12, or via catalysis by specific enzymes, such as adenosine deaminase or AMP deaminase. dITP can be generated by oxidative deamination of dATP, and incorporated into DNA10,13,14. In addition, hypoxanthine is a base moiety of inosine monophosphate (IMP), which is a normal intermediate metabolite in the de novo purine nucleotide metabolism pathway. Pang et al. reported a large increase of dI in DNA in strains of Escherichia coli, and Saccharomyces cerevisiae unable to 1 Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8581, Japan. 2Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8581, Japan. 3Research Center for Nucleotide Pool, Kyushu University, Fukuoka 812-8581, Japan. 4National Institute of Radiological Sciences, Chiba 263-8555, Japan. 5 Cancer Institute, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan. Correspondence and requests for materials should be addressed to D.T. (email: ) Scientific Reports | 6:32849 | DOI: 10.1038/srep32849 1 www.nature.com/scientificreports/ convert IMP to AMP or GMP, and unable to hydrolyze dITP/ITP15, suggesting the existence of a pathway from IMP, a normal nucleotide, to dI in DNA. Previous studies in mammalian cells have revealed that inosine triphosphatase (ITPA), encoded by the ITPA gene, hydrolyses inosine triphosphate (ITP) and dITP to IMP and dIMP with essentially the same efficiency16,17. Itpa knockout (KO) mice die before weaning with features of growth retardation and heart failure18. These results show that ITP and dITP are produced under physiological conditions in living cells, and that they induce vital dysfunction unless hydrolysed by ITPA. Furthermore, Itpa KO mouse embryos had increased levels of deoxyinosine/inosine in DNA/RNA, and primary mouse embryonic fibroblasts (MEFs) derived from Itpa KO embryos exhibited prolonged doubling time and increased chromosome abnormalities and accumulation of single-strand breaks (SSBs) in nuclear DNA compared with primary MEFs prepared from wild-type embryos19. We have previously performed a screen for ITP-binding proteins20 and revealed that nucleoside diphosphate linked moiety X-type motif16 (NUDT16), encoded by NUDT16, also hydrolyses (deoxy)inosine triphosphate and (deoxy)inosine diphosphate to (deoxy)inosine monophosphate. Knockdown of NUDT16 in either HeLa MR cells or ITPA-deficient MEF cells causes cell cycle delay in S phase, reduced cell proliferation, and increased accumulation of SSBs in nuclear DNA, suggesting that NUDT16, along with ITPA, has an important biological function in mammals as a sanitizing enzyme against inosine nucleotides. The human ITPA gene has a polymorphic variant, P32T, which has decreased enzymatic activity through three mechanisms: protein instability, decreased rate of catalysis, and improper mRNA splicing21–23. The P32T variant is associated with potentially severe adverse drug reactions towards the thiopurine drugs, azathioprine and 6-mercaptopurine24. Furthermore, the P32T variant is related to protection against adverse effects of Ribavirin treatment in patients with hepatitis C25–28. It has been reported that dI generated in DNA can be excised by several DNA repair systems in prokaryotes and eukaryotes. 3-Methyl-adenine DNA glycosylase II (AlkA) in Escherichia coli recognizes N-alkylpurine adducts, deaminated purine adducts, and lipid peroxidation-induced purine adducts in DNA, and cleaves N-glycosylic bonds within them. AlkA excises the hypoxanthine base from dI in DNA, forms an apurine/apyrimidine site (AP site) and initiates a base excision repair (BER) reaction29–31. Mammalian N-methylpurine-DNA glycosylase (MPG), which is known to excise at least 17 structurally diverse modified bases from DNA32, also removes (...truncated)