Nucleotide metabolism defects

The repair of stalled replication forks entails a coordinated transition from replication to recombination and back to replication. The recombination steps function to fill the DNA gap or rejoin the broken DNA branch to recreate the branched DNA structure at the replication fork. Lesions left behind in what is now duplex DNA are repaired by pathways such as base-excision or nucleotide-excision repair. Thus a wide range of enzymes encompassing every aspect of DNA metabolism ultimately take part in the repair of a stalled replication fork. This type of repair process is clearly a primary function of the homologous recombination system of every cell, and defects in recombinational DNA repair play an important role in human disease (Box 25-1). 

Pathways for the metabolic degradation of nucleotides are very important to the organism, as demonstrated by the fact that several genetic defects causing blocks in these pathways have serious consequences for the health of the organism. 

Overproduction of uric acid can occur due to excessive de novo purine synthesis, excessive dietary purines, or the conversion of tissue nucleic acid to purine nucleotides. When these purines are metabolized, the by-products are converted to uric acid by the enzyme xanthine oxidase. Increased levels of uric acid result if the overproduction exceeds excretion. Underexcretion of uric acid can be due to defects in the renal tubular mechanisms that regulate uric acid levels in the body, causing decreased filtration, decreased secretion, or increased reabsorption. 

The accumulation in blood and the excessive urinary excretion of orotic acid are postulated to result from the absence of either decarboxylase or the pyrophos-phorylase. These enzyme activities could not be directly assayed in the patient s tissues, but assays in the parents and two of the siblings indicated that the activities of both decarboxylase and phosphorylase were defective. In the patients described, the orotic aciduria and the megaloblastic anemia that it causes could be relieved by injecting nucleotides (cytidylic and uridylic acid). And in addition to its therapeutic significance, this observation also provides some invaluable information on the functioning of the pyrimidine metabolic pathway in vivo. 

Despite structural diversity in the superactive enzymes of individual families, studies of PRPP and purine metabolism carried out both vivo and in cells cultured from affected hemizygous males support the idea that a common mechanism accounts for the association of PRPP synthetase superactivity with uric acid overproduction. Increased intracellular PRPP concentrations and rates of PRPP generation as well as increased rates of all PRPP-dependent purine nucleotide synthetic processes are constant accompaniments of enzyme superactivity. These findings suggest a scheme to explain the association of the enzyme defect with uric acid overproduction PRPP synthetase superactivity -> increased intracellular PRPP generation and concentration > increased rate of purine nucleotide synthesis excessive uric acid synthesis. 

The number of inherited defects of the pyrimidine metabolism described so far is small, compared to that of the purine metabolism. Combined deficiency of orotate phosphoribosyltransferase (OPRT) (EC 2.4.2.10) and orotidine 5 -monophosphate decarboxylase (ODC) (EC 4.1.1.23), designated as type I hereditary orotic aciduria, presents with characteristic clinical features such as hypochromic anemia with a megaloblastic bone marrow and crystalluria. Only six patients have been described and, as far as we know, new cases have not been discovered recently. ODC deficiency with similar clinical phenomena and leading to increased urinary excretion of orotate and orotidine has been detected in only one patient (1). A third defect, a deficiency of pyrimidine 5 -nucleotidase (Py-5NX (EC 3.1.3.5.) in erythrocytes, is associated with chronic hemolytic anemia and prominent basophylic stippling of the erythrocytes due to accumulated pyrimidine nucleotides. An increasing number of patients have been reported, their detection being facilitated by the typical phenomena. We do not know whether the urinary pyrimidine profile in this condition is abnormal. 

Congenital defects and chemical inhibitors of these enzymes result in the accumulation of purine nucleosides and nucleotides which have been directly linked to impaired lymphocyte function and immunodeficiency syndromes (4,5). Since the uremic state is complicated by an increased susceptability to infection largely the result of acquired l3nnphocyte abnormalities, we have studied the ability of uremic erythrocytes (RBC) to metabolize vitro radiolabelled adenosine and deoxyadenosine utilizying a combind UV - radioactive high performance liquid chromatographic technique (HPLC) (1,2,3). 

The final steps of pyrimidine biosynthesis novo which are catalyzed by two sequential enzymes, orotate phosphoribosyltransfer-ase (OPRT) and orotidylic decarboxylase (ODC), involve the PP-ribose P dependent conversion of orotic acid to orotidine-5 -monophosphate (OMP) followed by decarboxylation at the 7 position to form uridine 5 -monophosphate (UMP) (Fig. 1). UMP is then utilized further in the synthesis of nucleic acids and co-enzymes. Defects at this site in this metabolic pathway are important for they can result in "pyrimidine starvation" from depletion of the intracellular pool of pyrimidine nucleotides. In man the rare genetic disease, orotic aciduria, involves a deficiency of both OPRT and ODC (Type 1) (Smith, Sullivan and Huguley, 1961) or, less commonly, only ODC (Type II) (Fox, 0 Sullivan and Firken, 1969). 

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