phosphorus-radioisotopes and deoxyguanosine-triphosphate

Genome replication of hepadnavirus proceeds by reverse transcription from a viral pregenomic RNA template by a virally encoded polymerase that possesses protein-priming, reverse transcriptase, DNA polymerase, and RNase H activities. Characterization of this enzyme has been hampered by failure to purify an active enzyme from virions and difficulties in expressing an active polymerase in heterologous systems. In this study, we constructed human hepatitis B virus polymerase cDNA under the control of a phage T7 promoter and expressed it in a rabbit reticulocyte lysate-coupled transcription-translation system. In vitro site-directed mutagenesis confirmed that the recombinant polymerase cDNA produced three products: a full-length protein (approximately 94 kDa), an internally initiated protein (approximately 81 kDa), and an N-terminal protein (approximately 40 kDa). The in vitro expressed polymerase possessed protein priming activity, as demonstrated by 32P-dGTP-labeling of the full size polymerase and the N-terminal portion of the molecule in an in vitro priming assay. The polymerase also exhibited polymerization activity, as detected in an in vitro polymerase assay by incorporation of radionucleotides into acid-precipitable polynucleotides and by synthesis of human hepatitis B virus (HBV) specific DNA with product lengths between 100 and 500 nucleotides. In addition, the polymerase was found to use an RNA sequence bearing HBV DR1/epsilon stem-loop motif as a template for DNA synthesis. Both the protein-priming and the reverse transcription activities of this recombinant polymerase are dependent on the RNA fragment containing the HBV DR1/epsilon stem-loop sequence known to be required for the polymerase activities. The in vitro systems used in this study will be applicable to further functional and biochemical studies of this enzyme.

Topics: Animals; Deoxyguanine Nucleotides; DNA-Directed DNA Polymerase; Hepatitis B virus; Humans; Phosphorus Radioisotopes; Protein Biosynthesis; Rabbits; Recombination, Genetic; Ribonuclease H; RNA-Directed DNA Polymerase; Templates, Genetic; Transcription, Genetic

We present a simple screening method for detecting a known point mutation, using only one 5'-biotinylated oligonucleotide primer, with its 3' end adjacent to the mutation site. In parallel reactions, an amplified DNA template encompassing the biotinylated oligonucleotide and mutation site undergoes 40 step-cycles of single nucleotide incorporation using Taq thermostable DNA polymerase and only one radioactive [alpha-32P]dNTP, specified by either the normal or mutant sequence. The oligonucleotides, now radioactively labelled at the 3' end according to the template sequence, are then trapped by streptavidin-coated magnetic beads, and the percent of radiolabel incorporated is determined directly by the Cerenkov method in a scintillation counter. The trapped-oligonucleotide nucleotide incorporation (TONI) assay has been used for the screening of a mitochondrial polymorphism, and has also been shown to distinguish the genotypes of hemoglobin A/C, A/A, A/S, and S/S. It is reproducible over at least a 100-fold range of radioisotope and a 10-fold range of oligonucleotide primer. This method is particularly useful for diagnosing mutations which do not produce alterations detectable by restriction enzyme analysis, since optimization of conditions is rarely necessary. In addition, it requires only a single oligonucleotide, and no electrophoretic separation of the allele-specific products. It thus represents an improved and simplified modification of the existing allele-specific primer extension methods (Kuppuswamy et al., Proc Natl Acad Sci USA 88:1143-1147, 1991; Sokolov, Nucl Acids Res 18:3671, 1989; Syvanen et al., Genomics 8:684-692, 1990).

Topics: Base Sequence; Deafness; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA, Mitochondrial; Genetic Testing; Hemoglobin A; Hemoglobin, Sickle; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Phosphorus Radioisotopes; Point Mutation; Polymerase Chain Reaction; Polymorphism, Genetic

We have introduced [alpha-32P]dGTP into permeabilized cells and measured the degree of methylation at CpG sites by nearest-neighbor analysis. This method reveals a lag of approximately 1 min between DNA synthesis and the modification event. When methylation is inhibited by the addition of S-adenosyl-L-homocysteine in the presence of continued DNA synthesis, the resulting hemimethylated sites are methylated immediately after the release of inhibition. The results suggest that the methylase activity in the cell allows immediate methylation but conditions at the replication fork bring about a short delay in the onset of the modification reaction.

Topics: Animals; Deoxyguanine Nucleotides; DNA; DNA Replication; Kinetics; L Cells; Methylation; Mice; Phosphorus Radioisotopes; S-Adenosylhomocysteine; S-Adenosylmethionine

We have found that 8-azidoadenosine 5'-triphosphate (8-azido-ATP) and its photolyzed product are competitive inhibitors of terminal deoxynucleotidyltransferase with respect to substrate deoxynucleoside triphosphates. A detailed characterization of the inhibitory effect of 8-azido-ATP revealed that its mechanism of inhibition is identical with that reported for ATP [Modak, M. J. (1978) Biochemistry 17, 3116-3120]. Photoactivation of the azido-ATP-enzyme complex results in the covalent binding of azido-ATP to terminal deoxynucleotidyltransferase. No significant incorporation of prephotolyzed azido-ATP or unsubstituted ATP into enzyme protein is noted when complexes of these nucleotides with enzyme were exposed to identical photoactivation conditions. The majority of incorporated analogue was associated with the 26 000-dalton subunit of terminal deoxynucleotidyltransferase. Incorporation of azido-ATP was further found to be absolutely dependent on the presence of a divalent cation. All four deoxyribonucleoside triphosphates as well as ATP and guanosine 5'-triphosphate were able to compete with azido-ATP during the incorporation experiment as judged by the competitive reduction in the cross-linking of the photoaffinity analogue to terminal deoxynucleotidyltransferase (TDT). In addition, substrate binding site directed inhibitors, pyrophosphate and pyridoxal 5'-phosphate, effectively blocked the incorporation of azido-ATP into enzyme protein, while several other inhibitors of TDT catalysis, namely, ethylenediaminetetraacetic acid, alpha, alpha'-dipyridyl, 1,10-phenanthroline, p-(chloromercuri)benzoate, Rose Bengal, and the presence of 0.5 M KCl, influenced the cross-linking reaction to varying degrees. A tryptic peptide analysis of the azido-ATP-labeled 26K subunit of TDT revealed that the majority of the incorporated photoaffinity analogue was present in two peptides.

Topics: Adenosine Triphosphate; Affinity Labels; Animals; Azides; Cattle; Deoxyguanine Nucleotides; DNA Nucleotidylexotransferase; DNA Nucleotidyltransferases; Kinetics; Phosphorus Radioisotopes; Photolysis; Thymus Gland

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