Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa- mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa- embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa- primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa- MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa- embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa- embryos. However, immortalized Itpa- MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa- MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.
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