A deoxyribonucleotidase in mitochondria: Involvement in regulation of dNTP pools and possible link to genetic disease

Abstract

Three cytosolic and one plasma membrane-bound 5-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5-and 2(3)-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith-Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease. Mitochondrial DNA synthesis occurs throughout the whole cell cycle, independent of nuclear DNA replication (1). It is catalyzed by a separate DNA polymerase that uses distinct 2Ј-deoxyribonucleoside 5Ј-triphosphate (dNTP) pools (2, 3), sequestered from cytosolic dNTPs by the mitochondrial membranes. What is the origin of mitochondrial dNTPs and how are pool sizes regulated? Despite considerable efforts, a mitochondrial ribonucleotide reductase has not been found, suggesting import of dNTPs or deoxyribonucleosides from the cytosol into mitochondria. dNTPs are synthesized by the cytosolic ribonucleotide reductase and can be imported directly by a permease of the mitochondrial membrane (4). Deoxyribonucleosides are derived from the extracellular fluid or by catabolism of dNTPs and, after import into mitochondria, phosphorylated by specific intramitochondrial deoxyribonucleoside kinases (5)(6)(7)(8).But what regulates the size of intramitochondrial dNTP pools? This is an important question, because it is well established for cytosolic dNTPs that pool imbalance is genotoxic (9) and can cause specific diseases. Thus, in some cases of hereditary severe immune deficiency, the accumulation of dATP (10, 11) or dGTP (12, 13) in the cytosol of blood cells results in the apoptotic destruction of B and͞or T cells. In a different autosomal recessive disease, neurogastrointestinal encephalomyopat...

Mutation analysis in 16 patients with mtDNA depletion

“…When the supply of deoxyribonucleotides exceeds the requirements for DNA replication, the surplus is degraded and leaves the cells. d -NT2 is a mitochondrial nucleotidase which d ephosphorylates specifically the 5'-and 2'(3')-phosphates of uracil and thymine deoxyribonucleotides (Rampazzo et al, 2000). In this respect, d-NT2 protects mitochondrial DNA replication from overproduction of dTTP.…”

Transgenic expression of the deoxynucleotide carrier causes mitochondrial damage that is enhanced by NRTIs for AIDS

“…In contrast, NRTI-sparing regimens caused no functional changes. The cellular processes of NRTI mitochondrial import and phosphorylation [16][17][18][19][20][21][22][23][24][25] received increasing interest and awareness as playing a role in the regulation of mtDNA replication. 17 The DNC TG here offered an approach to evaluate NRTI toxicity in vivo and linked NRTI toxicity to mitochondrial import and nucleotide homeostasis.…”

Real-time quantitative PCR assays for deoxycytidine kinase, deoxyguanosine kinase and 5’-nucleotidase mRNA measurement in cell lines and in patients with leukemia

“…27 Recently two new 5′-NTs have been 391 Table 3 Levels of mRNA expression of dCK, dGK, 5′-NT and the ratio of dCK and 5′-NT in pedriatic patients with leukemia reported of, one cytosolic 5′(3′)-deoxyribonucleotidase 28 and a mitochondrial 5′-nucleotidase, dNT-2. 29 Although the relative involvement of these nucleotidases in the catabolism of nucleoside analogues and their clinical relevance as prognostic factors remain to be studied. RQ-PCR and semi-quantitative PCR showed similar results for dGK.…”