Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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A mutator strain [AraCr (1.5)4], isolated from mutagenized cultures of multipotent mouse teratocarcinoma cells (embryonal carcinoma stem cells), exhibited a dNTP pool imbalance, with more than a 10-fold relative increase in the intracellular concentration of dCTP. The increase in the spontaneous rate of mutation for 6-thioguanine resistance was 3.6-fold and for ouabain resistance, 7.9-fold. Normalization of the dCTP/dTTP ratio by addition of thymidine and deoxycytidine to the media was associated with normalization of the mutation rates. AraCr (1.5)4 cell retained its multipotency (including chimerization potential) when injected into blastocysts. Moreover, its differentiated progeny expressed the dNTP pool imbalance and mutator phenotype in vitro. The preliminary finding of an increased frequency of morphologically abnormal embryos derived from a series of transplanted blastocysts injected with AraC2 (1.5)4 stem cells is consistent with significant phenotypic effects in vivo.
Somat Cell Mol Genet 1985 May
PMID:Multipotent mutator strain of mouse teratocarcinoma cells. 385 19

The intracellular deoxyribonucleoside triphosphate pools in mammalian cells affect diverse biological functions including the spontaneous or induced mutability. We have isolated from murine T-lymphosarcoma S49 cells, a mutant that is unable to convert dCMP to dUMP, contains deranged intracellular dNTP pools, and exhibits a mutator phenotype. The enzymatic defect in araC-6-1 cells is a deficiency of deoxycytidylate deaminase, which accounts for the high dCTP and low TTP intracellular pools. The addition of increasing concentrations of exogenous thymidine to araC-6-1 cells alters these dNTP pools in a predictable manner: increasing the TTP and diminishing the dCTP. Concomitant with this reversal of the dCTP:TTP ratio is a marked decrease in the mutation rate followed by an increase in the mutation rates at higher exogenous thymidine concentrations. This response of the mutation rate is in contrast to that seen in the control cell line containing normal deoxycytidylate deaminase. In the latter case, increasing thymidine concentration induces an enhanced mutation rate that parallels the later phase of the thymidine-induced mutation rate in araC-6-1 cells. The deficiency of deoxycytidylate deaminase, the endogeneous dNTP pool alterations, and the mutator phenotype of araC-6-1 cells are all recessive traits in cell-cell hybrids. These observations allow one to predict whether exogenous thymidine will be mutagenic, antimutagenic, or both for a given cell line and provide a basis for understanding conflicting reports in the literature concerning the effects of the thymidine on genomic stability.
Somat Cell Mol Genet 1985 Sep
PMID:The effects of exogenous thymidine on endogenous deoxynucleotides and mutagenesis in mammalian cells. 387 1

Nuclear and whole-cell deoxynucleoside triphosphate (dNTP) pools were measured in HeLa cells at different densities and throughout the cell cycle of synchronized CHO cells. Nuclei were prepared by brief detergent (Nonidet P-40) treatment of subconfluent monolayers, a procedure that solubilizes plasma membranes but leaves nuclei intact and attached to the plastic substratum. Electron microscopic examination of monolayers treated with Nonidet P-40 revealed protruding nuclei surrounded by cytoskeletal remnants. Control experiments showed that nuclear dNTP pool sizes were stable during the time required for isolation, suggesting that redistribution of nucleotides during the isolation procedure was minimal. Examination of HeLa whole-cell and nuclear dNTP levels revealed that the nuclear proportion of each dNTP was distinct and remained constant as cell density increased. In synchronized CHO cells, all four dNTP whole-cell pools increased during S phase, with the dCTP pool size increasing most dramatically. The nuclear dCTP pool did not increase as much as the whole-cell dCTP pool during S phase, lowering the relative nuclear dCTP pool. Although the whole-cell dNTP pools decreased after 30 h of isoleucine deprivation, nuclear pools did not decrease proportionately. In summary, nuclear dNTP pools in synchronized CHO cells maintained a relatively constant concentration throughout the cell cycle in the face of larger fluctuations in whole-cell dNTP pools. Ribonucleotide reductase activity was measured in CHO cells throughout the cell cycle, and although there was a 10-fold increase in whole-cell activity during S phase, we detected no reductase in nuclear preparations at any point in the cell cycle.
Mol Cell Biol 1985 Dec
PMID:DNA precursor pools and ribonucleotide reductase activity: distribution between the nucleus and cytoplasm of mammalian cells. 391 77

Isolation and characterization of Chinese hamster ovary cell mutants resistant to different DNA polymerase inhibitors (aphidicolin, ara-A and ara-C) have been described. A particular mutant (JK3-1-2A) characterized in detail was found to grow and synthesize DNA in medium containing an amount of aphidicolin tenfold greater than that which completely inhibited the growth and the DNA synthesis of the wild-type cells. An almost twofold increase in the specific activity of the DNA polymerase alpha was seen in this mutant. The mutant DNA polymerase showed altered aphidicolin inhibition kinetics of dCMP incorporation; the apparent Km for dCTP and the apparent Ki for aphidicolin were increased in the mutant. These alterations in the kinetic parameters were, however, abolished upon further purification of the enzyme. Ara-CTP was found to act as a competitive inhibitor of the dCMP incorporation by both the wild type and mutant enzymes. In contrast, the effect of aphidicolin on dCMP incorporation was either competitive (wild-type enzymes) or noncompetitive (mutant enzyme). The data presented showed that the sites of action for aphidicolin and ara-CTP were distinct; likewise the dCTP binding site appeared to be separate from other dNTP(s) binding sites. The drug resistance of the mutant was inherited as a dominant trait.
Mol Gen Genet 1985
PMID:Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors. I. Isolation and biochemical genetic characterization. 393 Sep 21

To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.
Mol Cell Biol 1985 Apr
PMID:Cloning of nascent monkey DNA synthesized early in the cell cycle. 399 Jun 92

Hydroxyurea and pyrimidine analogs have been shown to enhance the chemotherapeutic efficacy and the DNA excision repair-inhibitory capacity of arabinofuranosylcytosine (ara-C). Since various cell types are expected to respond differently to these combination treatments and since little is known about the nature of their antiproliferative effects, we have investigated the metabolism and uptake into acid-soluble pools of deoxycytidine (dCyd) and ara-C in cycling and non-cycling human diploid fibroblasts. A substantial fraction of dCyd is converted through deamination to deoxyuridine and thymidine nucleotides, and this occurs to a greater degree in log phase cultures. ara-C is more resistant to deamination and is metabolized primarily to ara-CTP. Hydroxyurea decreases the proportion of dCyd and ara-C which is deaminated under both growth conditions, leading to higher levels of ara-CTP and dCTP. Trifluorothymidine causes an accumulation of dUMP and decreases the formation of dCTP in log phase and confluent cells. Thymidine inhibits deamination in log phase cells but stimulates this pathway in noncycling cells. Dideoxythymidine did not appreciably alter the spectrum of metabolites of dCyd formed in log or confluent phase cells but was shown to inhibit the transport of dCyd and thymidine across the membrane. These studies provide information regarding the nature of the enhancement of the antiproliferative activity of ara-C by commonly used drugs and indicate that the cycling state of the target cell plays a major role in determining the metabolism of the nucleoside and the efficacy of chemotherapeutic treatments.
Mol Pharmacol 1985 Dec
PMID:Effects of hydroxyurea and thymidine derivatives on the uptake and metabolism of deoxycytidine and arabinofuranosylcytosine in log phase and contact-inhibited human diploid fibroblasts. 407 13

Mutants induced at the adenine phosphoribosyl transferase (aprt) locus by dTTP or dCTP pool imbalances were examined for alterations in genomic DNA sequences. No observable changes were detected by Southern blot analysis of most mutant DNAs, suggesting induction of base pair alterations or other events below our level of detection (approximately 30 base pairs). However, in a few strains (11 from a total collection of 125 mutant cell strains), we were able to localize these events to restriction endonuclease recognition sequences when the mutations resulted in the loss or gain of a particular site. The distribution of lost or gained sites in aprt-deficient mutants induced by the two types of pool imbalances clearly varied, with those occurring in a mutator strain with increased dCTP clustering at one end of the aprt gene. Mutants induced by dTTP also revealed novel events: multiple restriction site modifications in a small region of the aprt gene in one mutant and a small (approximately 50 base pairs) insertion or duplication of DNA sequences. As in previous studies, very few deletion or insertion mutants were detected at the aprt locus. The significance of these findings in terms of the known biochemical and genetic consequences of these pool imbalances is discussed.
Mol Cell Biol 1984 Sep
PMID:Structural alterations of the aprt locus induced by deoxyribonucleoside triphosphate pool imbalances in Chinese hamster ovary cells. 609 39

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4-6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653-664). Following a 30 s incubation with [3H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5' ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared with 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP, rUTP (0.1mM), as well as high levels of rATP (5mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.
 ...
PMID:The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells. 617 44

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.
Mol Cell Biol 1982 Mar
PMID:Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization. 618 Mar 4

In order to understand further the molecular mode of action of 5-Aza-2'-deoxycytidine (5-AZA-dCyd), a potent antileukemic agent, we prepared enzymatically 5-Aza-2'-deoxycytidine 5'-triphosphate (5-AZA-dCTP) and performed studies with purified DNA polymerase alpha and DNA methylase from mammalian cells. DNA polymerase alpha catalyzed the incorporation of 5-AZA-dCTP into DNA. The apparent Km value for 5-AZA-dCTP was estimated to be 3.0 microM; the Km of dCTP was 2.0 microM. The apparent Vmax of 5-AZA-dCTP was slightly lower than that for dCTP. 5-AZA-dCTP was a weak competitive inhibitor (Ki 4.3 microM) with respect to dCTP. Template studies with 5-AZA-dCTP showed that this nucleotide analogue was incorporated into poly(dIC), but not into poly(dAT), suggesting that the incorporation follows the rules of Watson-Crick base pairing. Incorporation of 5-AZA-dCTP into hemimethylated DNA produced a significant inhibition of DNA methylase. These results show that 5-AZA-dCTP is a very good substrate for DNA polymerase alpha and that its incorporation into DNA inhibits DNA methylation.
Mol Pharmacol 1983 Jul
PMID:Incorporation of 5-Aza-2'-deoxycytidine-5'-triphosphate into DNA. Interactions with mammalian DNA polymerase alpha and DNA methylase. 619 Nov 92


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