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

Three cytosolic and one plasma membrane-bound 5'-nucleotidases have been cl oned and characterized. Their various substrate specificities suggest widel y different functions in nucleotide metabolism. We now describe a 5'-nucleo tidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifica lly the 5'- and 2'(3')-phosphates of uracil and thymine deoxyribonucleotide s. 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 p rocessing during import into the mitochondrial matrix, The deduced amino ac id sequence is 52% identical to that of a recently described cytosolic deox yribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity, Mitochondrial localizati on of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cel ls expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the prep rotein lost the leader peptide. We suggest that dNT-2 protects mitochondria l DNA replication from overproduction of dTTP, in particular in resting cel ls. Mitochondrial toxicity of dTTP can be inferred from a severe inborn err or of metabolism in which the loss of thymidine phosphorylase led to dTTP a ccumulation and aberrant mitochondrial DNA replication, We localized the ge ne for dNT-2 on chromosome 17p11.2 in the Smith-Magenis syndrome-critical r egion, raising the possibility that dNT-2 is involved in the etiology of th is genetic disease.