Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Reverse transcription of the retroviral RNA genome begins with tRNA-primed synthesis of a minus-strand DNA, which subsequently acts as the template for the synthesis of plus-strand DNA. This plus-strand DNA is initiated at a unique location and makes use of a purine-rich RNA oligonucleotide derived by RNase H action on the viral RNA. To determine the variables that are relevant to successful specific initiation of plus-strand DNA synthesis, we have used nucleic acid sequences from the genome of Rous sarcoma virus along with three different sources of RNase H: avian myeloblastosis virus DNA polymerase, murine leukemia virus DNA polymerase, and the RNase H of Escherichia coli. Our findings include evidence that specificity is controlled not only by the nucleic acid sequences but also by the RNase H. For example, while the avian reverse transcriptase efficiently and specifically initiates on the sequences of the avian retrovirus, the murine reverse transcriptase initiates specifically but at a location 4 bases upstream of the correct site.
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PMID:Specificities involved in the initiation of retroviral plus-strand DNA. 168 26

S-Antigen (S-Ag) is a well characterized 45,000 m.w. photoreceptor cell protein. When injected into susceptible animal species, including primates, it induces an experimental autoimmune uveitis, a predominantly T cell-mediated autoimmune disease of the retina and uveal tract of the eye, and of the pineal gland. In this study we found an amino acid sequence homology between a uveitopathogenic site of S-Ag, several viral proteins and one additional nonviral protein. An experimental autoimmune uveitis and pinealitis was induced in Lewis rats with these different synthetic peptides, corresponding to the amino sequence of hepatitis B virus DNA polymerase, gag-pol polyprotein of Baboon endogenous virus and gag-pol polyprotein of AKV murine leukemia virus and potato proteinase inhibitor IIa, which contain three or more consecutive amino acids identical to peptide M in S-Ag. Lymph node cells from rats immunized with either peptide M or the different synthetic peptides showed a significant degree of cross-reaction. Mononuclear cells from monkeys (Macaca fascicularis) immunized with peptide M also showed significant proliferation when incubated with either peptide M or synthetic peptides as measured by in vitro lymphocyte mitogenesis assay using [3H]TdR. Based on our findings we conclude that a viral infection may sensitize the mononuclear cells that can cross-react with self proteins by a mechanism termed molecular mimicry. Tissue injury from the resultant autoantigenic event can take place in the absence of the infectious virus that initiated the immune response.
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PMID:Molecular mimicry between a uveitopathogenic site of S-antigen and viral peptides. Induction of experimental autoimmune uveitis in Lewis rats. 168 49

2',3'-Dideoxyuridine (ddUrd) exhibits poor if any anti-human immunodeficiency virus (HIV) activity in ATH8 and MT-4 cells. This is in agreement with the failure of ddUrd to be efficiently anabolized intracellularly to its 5'-triphosphate metabolite. However, 2',3'-dideoxyuridine-5'-triphosphate (ddUTP) proved to be a potent and selective inhibitor of the reverse transcriptase of HIV (Ki, 0.05 microM) and avian myeloblastosis virus (Ki, 1.0 microM). Bacterial DNA polymerase I, mammalian DNA polymerase alpha, terminal deoxyribonucleotidyl transferase, and Moloney murine leukemia virus reverse transcriptase were resistant to ddUTP. ddUTP is incorporated into the growing DNA chain principally at dTTP sites and inhibits further elongation. The potential of ddUTP as an anti-HIV therapeutic agent merits further investigation. However, to achieve this goal, it will be necessary to resort to techniques capable of delivering preformed phosphorylated ddUrd to the susceptible cells.
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PMID:Potent DNA chain termination activity and selective inhibition of human immunodeficiency virus reverse transcriptase by 2',3'-dideoxyuridine-5'-triphosphate. 168 52

The N syndrome is characterized by mental retardation, malformations, chromosome breakage, and development of T-cell leukemia (Opitz et al.: Proceedings of the II International Congress IASSMD pp 115-119, 1971; Hess et al.: Clinical Genetics 6:237-246, 1974b, American Journal of Medical Genetics [supplement] 3:383-388, 1987). N syndrome fibroblasts were studied to see if the high chromosome breakage rate associated with this apparently X-linked syndrome could be related to a deficiency of DNA polymerase alpha, a product of a gene localized to the X chromosome. Bleomycin, which is known to break double-stranded DNA, produced increased chromosome breakage in normal control, Fanconi anemia, and N syndrome fibroblasts. When aphidicolin was used to inhibit repair mediated by DNA polymerase alpha, both normal control and Fanconi anemia fibroblasts showed significantly more chromosome breakage than was produced by bleomycin alone, but there was no increase in the amount of breakage seen in the N syndrome fibroblasts over that seen with bleomycin alone. This suggests that the N syndrome is due to a mutation affecting the region of the X chromosome on which the gene for DNA polymerase alpha is located, and that the high risk of T-cell leukemia observed in the hemizygote is due to this DNA repair defect.
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PMID:DNA polymerase alpha defect in the N syndrome. 168 58

We have constructed a series of plasmids that, when introduced into Escherichia coli, induce the expression of high levels of either wild-type or mutated forms of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Mutant forms of RT that had been previously analyzed for their RNA-dependent DNA polymerase activity were tested for RNase H activity using an in situ polyacrylamide gel assay. Mutations affecting the RNase H are not clustered in a single region of the 66-kDa RT molecule. With only few exceptions, mutations that affect the RNase H activity also cause a substantial decrease in the DNA polymerase function. This suggests that, unlike the RT from murine leukemia virus (MuLV), it is difficult to genetically separate the catalytic domains responsible for the RNase H and DNA polymerase functions of HIV-1 RT. Those few mutations that differentially affect the RNase H and the polymerase activities of HIV-1 RT suggest that, as in MuLV, the polymerase domain is in the amino-terminus and the RNase H domain is in the carboxy-terminus. We have also generated chimeric molecules that are composed of sequences from the RT of HIV-1 and MuLV and these hybrid RTs were analyzed for their enzymatic properties. Two of these chimeric RTs possess RNase H activity but lack detectable DNA polymerase activity.
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PMID:Mutational analysis of the ribonuclease H activity of human immunodeficiency virus 1 reverse transcriptase. 169 64

The action of 3'-azido-3'-deoxythymidine 5'-triphosphate (N3dTTP) on DNA strand elongation catalyzed by human immunodeficiency virus type 1 reverse transcriptase was evaluated in comparison with human DNA polymerase alpha and proliferating cell nuclear antigen-independent DNA polymerase delta. Sequencing gel analysis demonstrated that the human immunodeficiency virus 1 reverse transcriptase preferentially incorporated N3dTTP into the T sites of the growing DNA strands and caused chain termination in a dose-dependent manner. This effect was observed even when the N3dTTP concentration was 0.3 microM, 100-fold less than dTTP. Studies with reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus showed that N3dTTP was also efficiently incorporated into DNA by these enzymes and terminated DNA strand elongation. In contrast, human DNA polymerases alpha and delta did not incorporate detectable amounts of N3dTTP into the DNA and were not inhibited by 300 microM N3dTTP. The selective incorporation of the chain-terminating nucleotide by the viral reverse transcriptases appears to be a molecular basis for the positive therapeutic index of 3'-azido-3'-deoxythymidine.
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PMID:Selective action of 3'-azido-3'-deoxythymidine 5'-triphosphate on viral reverse transcriptases and human DNA polymerases. 169 49

Within normal hemopoiesis, the intranuclear DNA polymerase TdT seems to be exclusively expressed by T and B lymphoid precursor cells. Double staining experiments showed that TdT can also be expressed in blast cells of certain acute myeloid leukemias. Recent reports described a very strong association between TdT expression and rearrangements of IgH and TcR genes in such AML specimens, suggesting a predominant lymphoid commitment of these TdT positive AML blasts. When submitting 24 serologically and morphologically well-characterized TdT positive AML specimens for additional genotypic analysis to determine the IgH and TcR gene configuration, we observed that only four had clonally rearranged IgH and/or TcR genes, whereas 20 had germ line configuration. This frequency is clearly lower than previously reported and not necessarily different from rearrangement frequencies reported for TdT negative AML (4-40%). It would seem to us, therefore, that the expression of TdT in otherwise well-defined AML blasts is not necessarily associated with a higher frequency of immunoglobulin and/or T cell receptor gene rearrangement.
Leukemia 1990 Apr
PMID:Terminal deoxynucleotidyl transferase and CD7 expression in acute myeloid leukemias are not associated with a high frequency of immunoglobulin and/or T cell receptor gene rearrangement. 169 41

Four flavonoids, 5,6,7-trihydroxyflavone (baicalein), 3,3',4',5,7-pentahydroxyflavone (quercetin), 3,3',4',5,6,7-hexahydroxyflavone (quercetagetin) and 3,3',4',5,5',7-hexahydroxyflavone (myricetin), were found to be potent inhibitors of reverse transcriptases from Rauscher murine leukemia virus (RLV) and human immunodeficiency virus (HIV). Under the reaction conditions employed, any one of these flavonoids almost completely inhibited the activity of RLV reverse transcriptase at a concentration of 1 microgram/ml. HIV reverse transcriptase was inhibited by 100%, 100%, 90% and 70% in the presence of 2 micrograms/ml quercetin, myricetin, quercetagetin and baicalein, respectively. The mode of inhibition of these flavonoids was competitive (RLV reverse transcriptase) or partially competitive (HIV reverse transcriptase) with respect to the template.primer complex, (rA)n.(dT), and noncompetitive with respect to the triphosphate substrate, dTTP. The Ki values for RLV reverse transcriptase were found to be 0.37 microM and 0.08 microM for baicalein and quercetin, respectively and those for HIV reverse transcriptase were 2.52 microM, 0.52 microM, 0.46 microM and 0.08 microM for baicalein, quercetin, quercetagetin and myricetin, respectively. Comparative studies with other flavonoids (hydroxyflavones, dihydroxyflavones and polyhydroxyflavones and flavanones) carried out to clarify the structure/activity relationships, revealed that the presence of both the unsaturated double bond between positions 2 and 3 of the flavonoid pyrone ring, and the three hydroxyl groups introduced on positions 5, 6 and 7, (i.e. baicalein) were a prerequisite for the inhibition of reverse transcriptase activity. Removal of the 6-hydroxyl group of baicalein required the introduction of three additional hydroxyl groups at positions 3, 3' and 4' (quercetin), to afford a compound still capable of inhibiting the reverse transcriptase activity. Quercetagetin which contains the structures of both baicalein and quercetin, and myricetin which has the structure of quercetin with an additional hydroxyl group on the 5' position also proved strong inhibitors of reverse transcriptase activity. The inhibition by baicalein of reverse transcriptase is highly specific, whereas quercetin and quercetagetin were also strong inhibitors of DNA polymerase beta and DNA polymerase I, respectively. Myricetin was also a potent inhibitor of both DNA polymerase alpha and DNA polymerase I.
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PMID:Differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular DNA and RNA polymerases. 169 72

We recently found that inhibition of MYB protein synthesis in human peripheral blood mononuclear cells (PBMC) exposed to human c-myb (designated MYB) antisense oligodeoxynucleotides prevents entry into S phase and cell proliferation. To determine the mechanism(s) by which down-regulation of human c-myb protein (MYB) synthesis interferes with DNA synthesis, we analyzed mRNA levels of DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), transcripts of two genes required for DNA synthesis, in normal and leukemic T lymphocytes exposed to MYB antisense oligodeoxynucleotides. Expression of DNA polymerase alpha was inhibited both in normal T lymphocytes progressing from G0 to S phase and in exponentially growing CCRF-CEM leukemic cells, whereas expression of PCNA was inhibited only in mitogen-stimulated PBMC and remained essentially unaffected in the leukemia T-cell line. The functional link between expression of MYB and DNA polymerase alpha mRNAs was further demonstrated by analyzing DNA polymerase alpha mRNA levels in a temperature-sensitive (ts) fibroblast cell line (TK-ts13; TK is thymidine kinase) constitutively expressing human MYB mRNA driven by the simian virus 40 (SV40) promoter. In the MYB-expressing TK-ts13 cells, DNA polymerase alpha mRNA levels were unaffected following shift to the nonpermissive temperature of 39.6 degrees C, whereas in the parental line, DNA polymerase alpha mRNA levels were readily down-regulated. These findings indicate that the expression of MYB is related to that of DNA polymerase alpha in cells expressing MYB at high levels and suggest that there is a functional link between c-myb and DNA polymerase alpha mRNA expression during cell cycle progression of normal T lymphocytes.
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PMID:Inhibition of T-cell proliferation by a MYB antisense oligomer is accompanied by selective down-regulation of DNA polymerase alpha expression. 169 13

Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with 4-(oxoacetyl)-phenoxyacetic acid (OAPA) results in the loss of DNA polymerase as well as template-primer binding activity but has no effect on the RT-associated RNase-H activity. Binding stoichiometry revealed that approximately 3 mol of OAPA bound per mole of enzyme, when complete enzyme activation occurred. However, in the presence of template-primer, OAPA does not abolish polymerase activity and 2 mol of OAPA remains bound to 1 mol of enzyme. This observation suggests that only one OAPA reactive site is responsible for the loss of polymerase activity. This site was located on a single tryptic peptide by comparing the maps of the native enzyme and the enzyme treated with OAPA in the presence and absence of template-primer. The appearance of a new peptide peak eluting at 125 min from a C-18 reverse-phase column was consistently noted in the tryptic digest of enzyme treated with OAPA. This peak was absent in tryptic peptides made from the control enzyme or the enzyme protein that was treated with OAPA in the presence of activated DNA or synthetic template-primers. Amino acid composition and sequence analyses of this peptide revealed that it spanned residues 312-342 in the primary amino acid sequence of MuLV RT. Since this peptide does not contain arginine residues and Lys-329 exhibited resistance to tryptic digestion, we conclude that Lys-329 is the target of OAPA action.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lysine-329 of murine leukemia virus reverse transcriptase: possible involvement in the template-primer binding function. 169 96


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