Abstract
5'-Nucleotidases are the catabolic members of the substrate cycles postulated to be involved in the regulation of intracellular deoxyribonucleoside triphosphate pools. Here, we attempt to identify the nature of the nucleotidases. Earlier, we constructed various mammalian cell lines that can be induced to overproduce the high K(m) 5'-nucleotidase (hkm-NT) or the 5'(3')-deoxynucleotidase (dNT-1). Now we labeled control and induced human 293 cells and hamster V79 cells with radioactive hypoxanthine or uridine and during a chase measured quantitatively the metabolism of ribo- and deoxyribonucleotides, DNA replication, and excretion of nucleosides into the medium. Overproduction of hkm-NT greatly increased excretion of inosine and guanosine but did not affect adenosine or deoxyribonucleosides. dNT-1 overproduction increased excretion of deoxycytidine, thymidine, and in particular deoxyuridine but also uridine and cytidine. We conclude that the hkm-NT is not involved in the regulation of deoxyribonucleotide pools but affects IMP and GTP pools. dNT-1, instead, appears to be the catabolic arm of substrate cycles regulating pyrimidine nucleotide pools.
Highlights
DNA replication and repair requires a continuous and balanced supply of the four deoxyribonucleoside triphosphates.1 Precursor pools are very small, suffice only for a few minutes of DNA replication in mammalian cells, and have to be continuously replenished during S phase [1]
The main results were as follows: (i) in all normal cells the bulk of newly synthesized purine and pyrimidine deoxyribonucleotides was incorporated into DNA, but a fraction was excreted into the medium as deoxyribonucleosides; (ii) inhibition of DNA synthesis greatly increased the excretion of deoxyribonucleosides, and inhibition of deoxyribonucleoside triphosphates (dNTPs) synthesis by hydroxyurea favored their import; and (iii) loss of thymidine kinase resulted in increased excretion of thymidine
The results suggest that the high Km 5-nucleotidase (hkm-NT) participates in the regulation of the pools of some purine ribonucleotides and that dNT-1 is involved in substrate cycles of pyrimidine ribo- and deoxyribonucleotides
Summary
DNA replication and repair requires a continuous and balanced supply of the four deoxyribonucleoside triphosphates (dNTPs). Precursor pools are very small, suffice only for a few minutes of DNA replication in mammalian cells, and have to be continuously replenished during S phase [1]. “Auxiliary” catabolic enzymes (deaminases, phosphorylases, and hydrolases) remove some of the nucleosides and thereby shift the equilibrium in the direction of catabolism (Fig. 1) The importance of these enzymes for the regulation of dNTP pools is apparent from the finding that their genetic loss leads to severe dysfunctions that depend on the accumulation of specific dNTPs in special cell types (reviewed in Ref. 5). The loss of either adenosine deaminase or purine nucleoside phosphorylase leads to severe combined immune deficiency caused by the accumulation of dATP and dGTP, respectively, in T and B cells These cells are low in nucleotidase activity [6], and the balance in their substrate cycles is normally shifted toward anabolism. Nucleotidases in Substrate Cycles and deoxyuridine, and loss of deoxycytidine kinase resulted in increased excretion of deoxycytidine and deoxyadenosine
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