Deoxynucleotide kinase

Bello, L.J. Bessman, M.J. The enzymology of virus-infected bacteria, IV. Purification and properties of the deoxynucleotide kinase induced by bacteriophage T2. J. Biol. Chem., 238, 1777-1787 (1963)... 

Adenylate kinase performs the essential function of recovering AMP formed by many enzymatic processes and converting it to ADP (Eq. 6-65) which can be reconverted to ATP by oxidative or substrate level phosphorylation. The enzyme is present in all organisms. In vertebrates different isoenzymes function in the cytosol, mitochondrial intermembrane space, and mitochondrial matrix.862 863 A group of other nucleotide and deoxynucleotide kinases convert nucleoside monophosphates into diphosphates.864 865 Some of them, e.g., uridylate kinase are similar in structure and properties to adenylate kinase.866 867 Another member of the adenylate kinase family is phosphoribulokinase, an important photosynthetic enzyme (see Fig. 17-14, step a).868... 

A deoxyribonuclease not normally present in E. coli is rapidly synthesized after infection with phage T5 (47). This DNase appears at approximately the same time as the other early phage-specific enzymes (DNA polymerase and deoxynucleotide kinase, etc.) induced following infection with this bacteriophage. 

The rllB region of T4 seems to be transcribed from a promoter to the left end of rllA and in addition from a promoter between rllA and rllB (Schmidt et al., 1970). A similar relationship with two promoters has been described for the T4 gene 1 (deoxynucleotide kinase Sakiyama and Buchanan,... 

Sakiyama, S., Buchanan, J. M. Relationship between molecular weight of T4 phage-induced deoxynucleotide kinase and the size of its messenger ribonucleic acid. J. biol. Chem. 248, 3150-3154 (1973). 

All NRTIs, as exemplified for AZT (Fig. 7), act in a similar fashion following their uptake by the cells, they are phosphorylated successively to their 5 -monophosphate, 5 -diphosphate, and 5 -triphosphate form (De Clercq 2002). Unlike the first phosphorylation step in the metabolic pathway of the acyclic guanosine analogues (see above), which is carried out by a virus-encoded enzyme (thymidine kinase), the first as well as the subsequent phosphorylations of the 2, 3 -dideoxynucleosides are carried out by cellular enzymes, that is, a 2 -deoxynucleoside (e.g., dThd) kinase, a 2 -deoxynucleotide (e.g., dTMP) kinase, and a (2 -deoxy)nucleoside 5 -diphosphate (NDP) kinase. 

Deoxynucleotides for DNA synthesis are made at the nucleoside diphosphate level and then have to be phosphorylated up to the triphosphate using a kinase and ATP. The reducing equivalents for the reaction come from a small protein, thioredoxin, that contains an active site with two cysteine residues. Upon reduction of the ribose to the 2 -deoxyri-bose, the thioredoxin is oxidized to the disulfide. The thioredoxin(SS) made during the reaction is recycled by reduction with NADPH by the enzyme thioredoxin reductase. 

Ribonucleotide reductase works on ribo-A, -U, -G, -C diphosphates to give the deoxynucleotide. The deoxyuridine, which is useless for RNA synthesis, is converted to deoxythymidine by the enzyme thymidylate synthase, which uses methylene tetrahydrofolate as a one-carbon donor. The odd thing here is that ribonucleotide reductase uses the UDP as a substrate to give the dUDP. This must then be hydrolyzed to the dUMP before thymidylate synthase will use it to make dTMP. Then the dTMP has to be kinased (phosphorylated) up to dTTP before DNA can be made. 

Many other enzymes can be used to remove or add groups to the ends of the DNA molecule. The three major ones are alkaline phosphatase, which removes a phosphate group from the 5 terminus polynucleotide kinase, which adds a phosphate group to a free 5 terminus and terminal deoxynucleotidyl transferase, which adds one or more deoxynucleotides to the 3 terminus. 

The deprotected oligonucleotide synthetic product is precipitated twice in ethanol, and a 0.5 fig/fd solution in water is prepared (concentration is measured from a UV absorption spectrum). One microliter of the oligo-deoxynucleotide solution is mixed with 2 fd of 10X PL, 5 fd of [y-32P]ATP (or [y-35S]ATP), 1 fd of T4 polynucleotide kinase, and 11 fd water. After incubation at 37 ° (for 45 min with [y-32P]ATP or for 2 hr with [y-35S]ATP), the reaction is stopped by the addition of 150 [A of 5 M ammonium acetate, pH 5.5, and 130 fd water and 10 fd of the yeast tRNA solution are added to the mixture before precipitation with 1 ml ethanol. After chilling at —70° for at least 15 min, the precipitate is collected by centrifugation (12,000 g, 15 min), redissolved, and submitted to two additional cycles of precipitation-redissolution. Finally, the precipitate is redissolved in 20 fd of gel loading mix and the mixture analyzed on a 8% acrylamide-7 Af urea slab gel in IX electrophoresis buffer, until the bromphenol blue has reached the middle of the gel. 

From Pharmacia MuMLV reverse transscriptase (FPLC pure), T4-polynucleotide kinase (FPLC pure), terminal deoxynucleotide transferase (TdT, FPLC pure). From Boehringer Mannheim E. coli DNA polymerase (endonuclease free), T4 DNA ligase (5 U/yl), T4 DNA polymerase. 

Severe combined immunodeficiency (SCID) due to adenosine deaminase deficiency (ADA) exhibits autosomal recessive inheritance, and may account for up to 209 of cases (l). Accumulation of intracellular toxic deoxynucleotides and/or S-adenosyl homocysteine particularly in T cells, is considered responsible for the severe lymphoid depletion and dysfunction observed in affected children (2). Unlike other forms of SCID, this variety is amenable to enzyme replacement therapy using regular fresh irradiated red blood cell transfusions as the source of enzyme. Another form of therapy suggested from in vitro studies, is the use of deoxycytidine which theoretically would act as a competitive substrate for deoxycytidine kinase, the enzyme considered responsible for the intracellular accumulation of deoxy-ATP (dATP). This study reports our experience of various treatments in three ADA deficient children. 

We have tentatively identified nanomolar accumulations of deoxyinosine in uremic plasma. This compound is directly toxic to lymphocyte activation. Toxicity could result from "trapping of deoxyinosine in lymphoid tissue by specific lymphoid deoxy kinases where it is phosphorylated to deoxynucleotides with resultant inhibition of DNA synthesis (9). Investigations are currently underway to examine the effect deoxynucleosides on deoxynucleotide formation in lymphocytes from uremic patients. 

Analogs of the natural deoxynucleoside triphosphates can be used for DNA synthesis, both in vivo and in vitro, but substitution of unnatural for natural deoxynucleotide must conform with the requirements for base-pairing in the Watson-Crick model of DNA. Thus with the purified enzyme system, thymine could be replaced by uracil or 5-bromouracil, 5-methyl- and 5-bromocytosine for cytosine, and hypoxanthine for guanine ( 6). Although chemically synthesized deoxyuridine triphosphate can be incorporated into DNA, there is apparently no kinase in nature which phosphorylates deoxyuridylate to the triphosphate stage. This may account for the absence of uracil nucleotides in DNA ( 6). 

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