phosphorus-radioisotopes and thymidine-5--triphosphate

Defects in mtDNA maintenance range from fatal multisystem childhood diseases, such as Alpers syndrome, to milder diseases in adults, including mtDNA depletion syndromes (MDS) and familial progressive external ophthalmoplegia (AdPEO). Most are associated with defects in genes involved in mitochondrial deoxynucleotide metabolism or utilization, such as mutations in thymidine kinase 2 (TK2) as well as the mtDNA replicative helicase, Twinkle and gamma polymerase (POLG). We have developed an in vitro system to measure incorporation of radiolabelled dNTPs into mitochondria of saponin permeabilized cells. We used this to compare the rates of mtDNA synthesis in cells from 12 patients with diseases of mtDNA maintenance. We observed reduced incorporation of exogenous alpha (32)P-dTTP in fibroblasts from a patient with Alpers syndrome associated with the A467T substitution in POLG, a patient with dGK mutations, and a patient with mtDNA depletion of unknown origin compared to controls. However, incorporation of alpha (32)P-dTTP relative to either cell doubling time or alpha (32)P-dCTP incorporation was increased in patients with thymidine kinase deficiency or PEO as the result of TWINKLE mutations compared with controls. The specific activity of newly synthesized mtDNA depends on the size of the endogenous pool diluting the exogenous labelled nucleotide. Our result is consistent with a deficiency in the intramitochondrial pool of dTTP relative to dCTP in cells from patients with TK2 deficiency and TWINKLE mutations. Such DNA precursor asymmetry could cause pausing of the replication complex and hence exacerbate the propensity for age-related mtDNA mutations. Because deviations from the normal concentrations of dNTPs are known to be mutagenic, we suggest that intramitochondrial nucleotide imbalance could underlie the multiple mtDNA mutations observed in these patients.

Topics: Cell Membrane Permeability; Deoxycytosine Nucleotides; Deoxyribonucleotides; DNA Helicases; DNA Polymerase gamma; DNA-Directed DNA Polymerase; DNA, Mitochondrial; Humans; Mitochondrial Diseases; Mitochondrial Proteins; Models, Biological; Mutation; Phosphorus Radioisotopes; Syndrome; Thymidine Kinase; Thymine Nucleotides

The light strand promoter of mammalian mitochondrial DNA gives rise to a primary transcript, but also to the RNA primer necessary for initiation of replication and 7S DNA synthesis as well as 7S RNA. Here we have studied the turnover of 7S DNA in isolated rat liver mitochondria and whether import of mitochondrial transcription factor A (mtTFA), which is necessary for transcription initiation, increases its rate of synthesis. 7S DNA was present as two species, probably due to two different sites of RNA-DNA transition. Time course and pulse-chase experiments showed that the half-life of this DNA is approximately 45 min. Import of mtTFA, produced in vitro, into the mitochondrial matrix in stoichiometric amounts significantly increased the rate of 7S DNA formation. We conclude that isolated rat liver mitochondria faithfully synthesize and degrade 7S DNA and that increased matrix levels of mtTFA are sufficient to increase its rate of synthesis, strongly supporting the hypothesis that this process is transcription primed.

Topics: Animals; Biological Transport; DNA Replication; DNA-Binding Proteins; DNA, Mitochondrial; Male; Mitochondria, Liver; Mitochondrial Proteins; Nuclear Proteins; Phosphorus Radioisotopes; Rats; Rats, Wistar; Thymine Nucleotides; Time Factors; Transcription Factors

Topics: 3T3 Cells; Animals; Blood; Blotting, Northern; Cell Cycle; Cell Line; Cell Nucleus; Cloning, Molecular; Culture Media, Serum-Free; Culture Techniques; DNA; DNA Probes; DNA, Complementary; Enzyme Induction; Genes, fos; GTP Phosphohydrolases; Indicators and Reagents; Mice; Phosphorus Radioisotopes; Radioisotope Dilution Technique; rho GTP-Binding Proteins; Thymidine; Thymine Nucleotides; Transcription Factors; Transcription, Genetic

Topics: Animals; Cattle; DNA; DNA Polymerase I; DNA Polymerase III; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Isotope Labeling; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Substrate Specificity; Templates, Genetic; Thymine Nucleotides; Viral Proteins

Topics: Animals; Bacteriophage T7; Base Sequence; Binding Sites; Cattle; DNA Primase; DNA Primers; DNA Replication; DNA-Directed DNA Polymerase; Indicators and Reagents; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Recombinant Proteins; RNA Nucleotidyltransferases; Thymine Nucleotides; Thymus Gland; Tritium; Zinc

Topics: Bacterial Proteins; Cell-Free System; Chromosomes, Bacterial; Deoxyribonucleotides; DNA Replication; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerases; DNA, Bacterial; Escherichia coli; Kinetics; Phosphorus Radioisotopes; Plasmids; Radioisotope Dilution Technique; Replication Origin; Ribonuclease H; Templates, Genetic; Thymine Nucleotides

The repair of damage induced in DNA by ultraviolet light involves excision of the damage and then repair synthesis to fill the gap. We investigated the sites of repair synthesis using MRC-5 fibroblasts and HeLa cells in G1 phase. Cells were encapsulated in agarose microbeads to protect them during manipulation, irradiated, incubated to allow repair to initiate, and permeabilized with streptolysin O to allow entry of labelled triphosphates; [32P]dTTP was incorporated into acid-insoluble material in a dose-dependent manner. Incubation with biotin-16-dUTP allowed sites of incorporation to be indirectly immunolabeled using a FITC-conjugated antibody; sites were not diffusely spread throughout nuclei but concentrated in discrete foci. This is similar to sites of S phase activity that are attached to an underlying nucleoskeleton. After treatment with an endonuclease, most repaired DNA electroeluted from beads with chromatin fragments; this was unlike nascent DNA made during S phase and suggests that repaired DNA is not as closely associated with the skeleton. However, the procedure destroyed repair activity, so repaired DNA might be attached in vivo through a polymerase that was removed electrophoretically. Therefore this approach cannot be used to determine decisively whether repair sites are associated with a skeleton in vivo.

Topics: Cell Cycle; Cell Line; Cell Nucleus; DNA; DNA Damage; DNA Repair; Dose-Response Relationship, Radiation; G1 Phase; HeLa Cells; Humans; Kinetics; Phosphorus Radioisotopes; Thymine Nucleotides; Ultraviolet Rays

The repair of damage induced in DNA by ultraviolet light involves excision of the damaged sequence and synthesis of new DNA to repair the gap. Sites of such repair synthesis were visualized by incubating permeabilized HeLa or MRC-5 cells with the DNA precursor, biotin-dUTP, in a physiological buffer; then incorporated biotin was immunolabeled with fluorescent antibodies. Repair did not take place at sites that reflected the DNA distribution; rather, sites were focally concentrated in a complex pattern. This pattern changed with time; initially intense repair took place at transcriptionally active sites but when transcription became inhibited it continued at sites with little transcription. Repair synthesis in vitro also occurred in the absence of transcription. Repair sites generally contained a high concentration of proliferating cell nuclear antigen but not the tumour-suppressor protein, p53.

Topics: Amanitins; Cell Line; Cell Membrane Permeability; Cell Nucleus; DNA; DNA Damage; DNA Repair; HeLa Cells; Humans; Kinetics; Phosphorus Radioisotopes; Proliferating Cell Nuclear Antigen; Thymine Nucleotides; Transcription, Genetic; Tumor Suppressor Protein p53; Ultraviolet Rays

In order to further define the enzymatic properties of yeast DNA polymerase delta, the Saccharomyces cerevisiae POL3 gene, whose expression is highly toxic to bacteria in most cloning vectors, was cloned into a new T7 expression vector (W. C. Brown and J. L. Campbell, submitted for publication) which allowed efficient overexpression in bacteria. Fifteen mg of polymerase were obtained from 3 g of cells. Since the protein is produced in insoluble form, to obtain active polymerase, inclusion bodies were solubilized with urea. DNA polymerase delta (124 kDa) was purified in the presence of urea and then renatured by dialysis against buffers containing decreasing concentrations of urea. Optimal protein concentration for refolding was 5 micrograms/ml. By several criteria the enzyme obtained is comparable with that from yeast: specific activity, electrophoretic mobility, template preference, sensitivity to inhibitors, and processivity. The electrophoretic mobility suggests that, unlike DNA polymerase alpha, polymerase delta is not posttranslationally modified in yeast. Polyclonal antibody was raised against the full-length DNA polymerase delta from bacteria and shown to cross-react with the protein purified from yeast on protein blots. The renatured protein also exhibits an exonucleolytic activity. Further examination of this nuclease determined it to be a 3' to 5' exonuclease with the characteristics of a proofreading activity. The presence of this nuclease in the highly purified bacterial polymerase provides biochemical confirmation of earlier genetic evidence (Simon, M., Giot, L., and Faye, G. (1991) EMBO J. 10, 2165-2170) that suggested that DNA polymerase delta's core catalytic subunit contains an intrinsic 3' to 5' exonuclease.

Topics: Aphidicolin; Cloning, Molecular; Deoxyribonucleotides; DNA Polymerase III; DNA-Directed DNA Polymerase; DNA, Fungal; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Escherichia coli; Exonucleases; Kinetics; Molecular Weight; Phosphorus Radioisotopes; Protein Denaturation; Recombinant Proteins; Restriction Mapping; Saccharomyces cerevisiae; Thymine Nucleotides; Tritium

The distribution of primer RNA and RNA-primed nascent DNA in nuclei of CCRF-CEM leukemia cells was examined, and the primer RNA purified from the nuclear matrices of these cells was characterized. RNA-primed nascent DNA was radiolabeled by incubating whole-cell lysates with [alpha-32P]ATP and [3H]dTTP in the presence of approximately physiological concentrations of the remaining ribo- and deoxyribonucleoside triphosphates. The primer RNA was purified by cesium chloride density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis. Nuclear subfractionation studies revealed that at least 94% of the primer RNA and RNA-primed nascent DNA were located within the insoluble matrix fraction of the nucleus. The predominant primer RNA isolated from the nuclear matrix was 8-10 nucleotides in length, and several lines of evidence indicated that this oligoribonucleotide was the functional primer RNA. Essentially all of the matrix primer RNA was covalently linked to the newly replicated DNA as demonstrated by its buoyant density in cesium chloride gradients, phosphate-transfer analysis, and sensitivity to DNase I. Analysis of 32P transfer from [alpha-32P]dTTP revealed a random distribution of ribonucleotides at the 3'-end of the primer RNA. Data obtained from mixing experiments indicated that the association of RNA-primed nascent DNA with the nuclear matrix was not the result of aggregation of these fragments with the nuclear matrix. No significant amount of either primer RNA, RNA-primed nascent DNA, or phosphate transfer was detected in the high-salt-soluble (nonmatrix) fraction of the nucleus, although the nonmatrix fraction contained most of the newly replicated DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Autoradiography; Cell Line; Centrifugation, Density Gradient; DNA, Neoplasm; Electrophoresis, Polyacrylamide Gel; Humans; Kinetics; Molecular Weight; Phosphorus Radioisotopes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Radioisotope Dilution Technique; RNA, Neoplasm; Thymine Nucleotides; Tritium; Tumor Cells, Cultured

Subunit B1 of Escherichia coli ribonucleotide reductase contains one type of allosteric binding site that controls the substrate specificity of the enzyme. This site binds the allosteric effector dTTP as well as other nucleoside triphosphates. Cross-linking of dTTP to protein B1 by direct photoaffinity labeling, as well as the isolation and sequence determination of the labeled tryptic peptide, has recently been reported [Eriksson, S., Sjöberg, B.-M., Jörnwall, H., & Carlquist, M. (1986) J. Biol. Chem. 261, 1878-1882]. In this study, we have further purified the dTTP-labeled peptide and characterized it using UV spectroscopy. Two types of dTTP-cross-linked peptide were found: one having an absorbance maximum at 261 nm typical for a dTTP spectrum, i.e., containing an intact 5,6 double bond, and one minor form with low absorbance at 261 nm. In both cases, the same amino acid composition was found, corresponding to the peptide Ser291-X-Ser-Gln-Gly-Gly-Val-Arg299 in the B1 sequence with X being Cys-292 cross-linked to dTTP. Isotope labeling experiments revealed that one proton in the 5-methyl group of thymine was lost during photoincorporation. Therefore, the cross-linking occurs via the 5-methyl group to Cys-292 in a majority of incorporated dTTPs, but a second, possibly 5,6-saturated form of incorporated nucleotide was also detected. The reasons for the high stereospecificity of the reaction and the possible structure of the allosteric site of protein B1 are discussed.

Topics: Affinity Labels; Allosteric Site; Escherichia coli; Guanosine Diphosphate; Macromolecular Substances; Phosphorus Radioisotopes; Ribonucleotide Reductases; Thymine Nucleotides; Tritium; Ultraviolet Rays

Current evidence suggests that distinct mechanisms exist to regulate precursor synthesis for nuclear and mitochondrial DNA replication. We tested this is mouse L cells by asking whether nuclear and mitochondrial DNAs become labeled to equivalent specific activities when provided with an exogenous nucleic acid precursor. Cells were grown in [32P]orthophosphate-containing medium long enough to bring all pools to equivalent specific activities. [6-3H]Uridine was added to the medium as a general pyrimidine precursor. At intervals, cells were harvested and nuclear and mitochondrial DNA was isolated. After enzymatic hydrolysis of each DNA fraction to deoxyribonucleoside 5'-monophosphates, these were separated by high performance liquid chromatography and the 3H/32P ratio in each pyrimidine was determined as an index of the specific activity of DNA pyrimidine residues. The dTMP residues in nuclear and mitochondrial DNA reached roughly equal specific activities and at comparable rates. However, dCMP residues in mitochondrial DNA reached maximal specific activities more rapidly than those in nuclear DNA, and the maximal values attained were nearly twice those seen either with the nuclear DNA dCMP residues or in the dTMP residues from either DNA. This indicates that the pathways leading to dCTP synthesis are organized so that mitochondria can use exogenous precursors more effectively than can the nucleus. The nature of this compartmentation is not clear, but it evidently involves one or more steps beyond the divergence point between pathways to dCTP and dTTP.

Topics: Animals; Cell Compartmentation; Cell Nucleus; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA; L Cells; Mice; Mitochondria; Phosphorus Radioisotopes; Thymine Nucleotides; Tritium; Uridine