Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A series of antisera directed against amino acid sequences from different segments of the duck hepatitis B virus (DHBV) P-gene were shown to immunoprecipitate DHBV DNA molecules that were covalently linked to the DHBV DNA terminal protein. Restriction analysis and sizing after protease treatment demonstrated that the P-gene proteins were bound to the 5'-end of the DHBV DNA minus-strand which was mapped to a G-residue in the centre of the repeat sequence DR1. Resistance to alkali treatment indicated a phosphodiester linkage to tyrosine between protein and DNA. Limited protease treatment prior to immunoprecipitation cleaved C-terminal P-proteins from the viral DNA, indicating that the terminal protein forms a separate domain encoded in the N-terminal part of the P-gene. Functional analysis of a deletion mutant confirmed the notion that a non-essential spacer separates the terminal protein from the polymerase domain residing in the C-terminal half of the P-gene. Thus, the major proteins required for hepadnaviral reverse transcription, namely the primer, DNA polymerase, and possibly also RNase H, appear to be synthesized as a polyprotein precursor which is at least initially linked as such to its first DNA product.
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PMID:The amino-terminal domain of the hepadnaviral P-gene encodes the terminal protein (genome-linked protein) believed to prime reverse transcription. 285 56

We characterized a number of important features of the structure of the cohesive overlap region of the DNA genome of duck hepatitis B virus. The 5'-terminal nucleotide of minus-strand DNA was localized to nucleotide 2537, a G residue within the 12-base repeat sequence DR1. This G residue was shown to be the site of a covalent linkage to a protein, consistent with speculation that this protein is the primer of minus-strand synthesis, which occurs by reverse transcription. The 3' terminus of the minus strand was heterogeneous, being mapped to nucleotides 2530 and 2531, indicating that the minus strand is terminally redundant by seven or eight bases and ends at the putative 5' end of the transcribed RNA template (pregenome) for reverse transcription. We previously demonstrated that the presumptive RNA primer of plus-strand synthesis remains attached to plus-strand DNA during virus maturation; moreover, the sequence of this primer suggested an origin from the 5' end of the pregenome (J.-M. Lien, C. E. Aldrich, and W. S. Mason, J. Virol. 57:229-236, 1986). We show here that over 75% of plus-strand primers are capped, further supporting the idea that these primers are uniquely derived from the 5' end of the pregenome. Finally, we found that seemingly mature duck hepatitis B virus genomes are incomplete by at least 12 bases, in that the 12-base repeat sequence DR2 is not copied into plus-strand DNA during virus maturation. Since DR2 in virion DNA is duplexed with the RNA primer of plus-strand synthesis, it is possible that the failure to make complete plus strands is due to an inability of the viral DNA polymerase to carry out a displacement of the bound RNA primer.
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PMID:Initiation and termination of duck hepatitis B virus DNA synthesis during virus maturation. 368 60

Hepadnaviruses employ a unique mechanism for the initiation of RNA-directed DNA synthesis. Initially, four bases (5'-GTAA-3') are added to a tyrosine residue of the viral polymerase by reverse transcription of a bulge sequence in epsilon, a stem-loop structure which functions as the packaging signal for pregenomic RNA. This protein-DNA complex acts as the primer for minus-strand elongation from the 3' sequence, DR1. To understand this process in greater detail, we investigated whether the protein-mediated priming of viral DNA synthesis is affected by nucleotide analogs. By using cell-free expression of duck hepatitis B virus (DHBV) reverse transcriptase (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992), the 5'-triphosphate of the thymidine analog fialuridine (FIAU) was shown to inhibit the incorporation of radiolabeled TMP into primer DNA in a dose-dependent manner. Inhibition by the 5'-triphosphate of FIAU (FIAU-TP) was nearly complete at a concentration of 10 microM. The dideoxynucleotide analogs ddGTP, ddTTP, and 3'-azidodeoxythymidine triphosphate, known inhibitors of DHBV endogenous DNA polymerase, did not affect substantially the synthesis of primer DNA. Alternate substrate analysis suggested that FIAU is incorporated efficiently into nascent primer DNA as an analog of thymidine. Using site-directed mutagenesis to construct a mutant RNA template yielding a primer with the sequence 5'-GTAC-3', we demonstrated that FIAU-TP inhibited the incorporation of TMP, had no effect on that of dAMP, and decreased markedly the incorporation of dCMP. These results show that the synthesis of full-length DHBV primer DNA is inhibited by FIAU-TP but not by the dideoxynucleotide analogs that we tested. The significance of these findings as they relate to HBV DNA replication is discussed.
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PMID:Priming of duck hepatitis B virus reverse transcription in vitro: premature termination of primer DNA induced by the 5'-triphosphate of fialuridine. 752 86

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.
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PMID:Expression of an enzymatically active polymerase of human hepatitis B virus in an coupled transcription-translation system. 1043 46

Different mechanisms have been proposed to account for mitochondrial DNA (mtDNA) instability based on the presence of short homologous sequences (direct repeats, DR) at the potential boundaries of mtDNA rearrangements. Among them, slippage-mispairing of the replication complex during the asymmetric replication cycle of the mammalian mitochondrial DNA has been proposed to account for the preferential localization of deletions. This mechanism involves a transfer of the replication complex from the first neo-synthesized heavy (H) strand of the DR1, to the DR2, thus bypassing the intervening sequence and producing a deleted molecule. Nevertheless, the nature of the bonds between the DNA strands remains unknown as the forward sequence of DR2, beyond the replication complex, stays double-stranded. Here, we have analyzed the base composition of the DR at the boundaries of mtDNA deletions and duplications and found a skewed pyrimidine content of about 75% in the light-strand DNA template. This suggests the possible building of a DNA triple helix between the G-rich neo-synthesized DR1 and the base-paired homologous G.C-rich DR2. In vitro experiments with the purified human DNA polymerase gamma subunits enabled us to show that the third DNA strand may be used as a primer for DNA replication, using a template with the direct repeat forming a hairpin, with which the primer could initiate DNA replication. These data suggest a novel molecular basis for mitochondrial DNA rearrangements through the distributive nature of the DNA polymerase gamma, at the level of the direct repeats. A general model accounting for large-scale mitochondrial DNA deletion and duplication is proposed. These experiments extend to a DNA polymerase from an eucaryote source the use of a DNA triple helix strand as a primer, like other DNA polymerases from phage and bacterial origins.
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PMID:Base composition at mtDNA boundaries suggests a DNA triple helix model for human mitochondrial DNA large-scale rearrangements. 1208 9