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:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The spatial and temporal relationship between the polymerase and RNase H activities of human immunodeficiency virus type 1 reverse transcriptase has been examined by using a 40-mer RNA template and a series of DNA primers of lengths ranging from 15 to 40 nucleotides, hybridized to the RNA, as substrates. The experiments were executed in the absence and presence of heparin, an efficient trap to sequester any free or dissociated reverse transcriptase, thus facilitating the study of events associated with a single turnover of the enzyme. The results indicate a spatial separation of 18 or 19 nucleotides between the two sites. To examine the effect of concomitant polymerization on the RNase H activity, the substrate was doubly 5' end labeled on the RNA and DNA. This enabled the study of RNase H activity as a function of polymerization in a single experiment, and the results in the absence and presence of heparin indicate a tight temporal coupling between the two activities.
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PMID:Human immunodeficiency virus type 1 reverse transcriptase: spatial and temporal relationship between the polymerase and RNase H activities. 127 94

We have examined the RNA-dependent and DNA-dependent polymerase and ribonuclease H catalytic activities of human immunodeficiency virus reverse transcriptase using rapid transient kinetic methods with defined synthetic 25/45-mer DNA/RNA and DNA/DNA primer/templates. The Kd value for interaction of the enzyme with duplex DNA was 4.7 nM, and the value for RNA/DNA heteroduplex was of similar magnitude. A pre-steady state burst of nucleoside triphosphate incorporation was observed for both DNA and RNA templates. Analysis of the dATP concentration dependence of the burst rate provided Kd values for dATP of 4 and 14 microM and maximum rates of single nucleotide incorporation, kpol, of 33 and 74 s-1, for DNA and RNA templates, respectively. Subsequent turnovers were limited by the rate of dissociation of the primer/template from the enzyme at rates of 0.18 and 0.06 s-1 for duplex DNA and RNA/DNA heteroduplex, respectively. Analysis of rates of DNA polymerization and RNA cleavage using the RNA template revealed that the two activities are independent of one another. The polymerization rate (4-70 s-1) was dependent on dATP concentration, whereas the RNA cleavage occurred at a constant rate of 10 s-1 over the 100-fold dATP concentration range (2-200 microM). Examination of the RNA cleavage products resulting from a single turnover indicates that the polymerase and ribonuclease domains of the enzyme are separated by a distance corresponding to 19 bases of RNA/DNA heteroduplex, consistent with the recently published crystal structure (Kohlstaedt, L. A., Wang, J., Friedman, J., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Analysis of the kinetics of processive synthesis suggested that the initial binding of dNTP leads to a faster rate of dissociation of DNA from the enzyme. Further investigation supported a two-step dNTP binding mechanism with the formation of an initial E.DNA.dNTP complex followed by a more stable E'.DNA.dNTP complex. The Kd values for incorporation of incorrect nucleoside triphosphates opposite a DNA template thymidine were 1010 microM for dGTP, 1240 microM for dCTP, and 840 microM for dTTP. The corresponding maximum kpol rates were 4.8 s-1 for dGTP, 0.52 s-1 for dCTP, and 0.41 s-1 for dTTP. These values provide fidelity estimates of 1740 for discrimination against dGTP, 19,700 for dCTP, and 16,900 for dTTP misincorporations at this site.
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PMID:Mechanism and fidelity of HIV reverse transcriptase. 128 79

Inhibition of polypeptide chain elongation with the mRNA-complementary (antisense) oligonucleotide has been realized through a RNase H independent mechanism. Nuclease resistant complementary non-natural alpha-17-mer oligonucleotide did not inhibit cell-free protein biosynthesis of beta-globin in the wheat germ system because it did not elicit RNase H activity. Linkage of alkylating group [4-(N-2-chloroethyl-N-methyl)-aminobenzyl]-methylamine to the 5'-terminus of the alpha-oligomer led to the formation of its covalent adduct with mRNA which could not be translated in vitro. Linkage of hydrophobic residues to the terminal phosphates of natural oligonucleotides increased their stability against nucleases and uptake by human cancer cells. A porphyrin, substituted in the meso-position by aromatic groups, gave a rise to an approximately six-fold increase of uptake and cholesterol a 30-100-fold increase. Eighty percent of bound derivatives were found in cytoplasmic cellular fractions.
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PMID:Effect of the terminal phosphate derivatization of beta- and alpha-oligodeoxynucleotides on their antisense activity in protein biosynthesis, stability and uptake by eucaryotic cells. 132 80

A sequence of the rabbit alpha-globin mRNA is the primary target for ODN1, an unmodified 15-nucleotide (nt) antisense oligodeoxyribonucleotide (oligo). ODN1 prevented in vitro translation of both alpha- and beta-globin mRNAs in wheat germ extract. Nine secondary sites exhibiting more than 60% complementarity with ODN1 were present in the beta-globin message. The ODN1 inhibition of beta-globin synthesis was shown to be mediated by RNase H cleavage of the beta-globin mRNA at three partially complementary sites. Sandwich-type oligos consisting of a stretch of unmodified nt with a few methylphosphonate residues at both 5' and 3' ends were derived from ODN1. We have demonstrated that one such analogue (ODN2), with five phosphodiester linkages in the central region, exhibited improved specificity for alpha-globin mRNA compared with the unmodified parent 15-mer, due to a reduced ability of RNase H to cleave beta-mRNA/ODN2 mismatched duplexes.
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PMID:RNase H-mediated inhibition of translation by antisense oligodeoxyribonucleotides: use of backbone modification to improve specificity. 133 11

A synthetic RNA oligonucleotide (15-mer) corresponding to the 3' end of the lysine tRNA primer was hybridized to single-stranded DNA containing the human immunodeficiency virus type 1 (HIV-1) primer-binding site and extended with a DNA polymerase. The resulting structures were used to study primer removal by the RNase H activity of HIV-1 reverse transcriptase. The initial cleavage event removes the RNA primer as a 14-mer and leaves a single ribonucleotide A residue bound to the 5' end of the DNA strand. This result explains the observation by several groups that HIV-1 circle junctions contain 4 bp that are not present in the integrated provirus instead of the predicted 3 bp. Subsequent cleavage events occur at other sites internal to the RNA molecule, and the ribonucleotide A residue on the end of the DNA strand is ultimately removed. Therefore, the biologically relevant cleavage that produces the 14-mer reflects the kinetics of the reaction as well as a specificity for nucleic acid sequence. When the RNA oligonucleotide alone was hybridized to the primer-binding site and tested as a substrate for HIV-1 RNase H, the cleavage pattern near the 3' end of the RNA was altered.
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PMID:Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase. 137 87

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is one of the main targets in approaches to the chemotherapy of AIDS. A detailed knowledge of structure-function relationships of this enzyme is a prerequisite for rational drug design. We have used monoclonal antibodies as tools to identify functionally important regions of the protein. The preparation of 23 murine monoclonal antibodies (mAb) against HIV-1 reverse transcriptase and their different effects on the enzyme are described. The interaction of purified mAbs with HIV-1 RT was demonstrated by enzyme-linked immunosorbent assay (ELISA), Western blots, and high performance liquid chromatography size exclusion chromatography. One of the antibodies also recognized recombinant HIV-2 RT. Antibody binding epitopes on HIV-1 RT were analyzed by immunoblotting using cyanogen bromide fragmented RT, C-terminally truncated mutants, and a peptide ELISA employing 15-mer synthetic overlapping peptides spanning nearly the complete polypeptide chain. The epitopes were mapped within three domains corresponding to amino acids 200-230, 300-428, and 528-560. Two mAbs show neutralizing properties on enzymatic functions of RT. One affects the polymerase activity and to a certain degree the RNase H activity of the enzyme, whereas the other inhibits the latter activity exclusively. mAb 28, which blocks the polymerase activity, interferes with the nucleotide binding region of RT, as shown by fluorescence spectroscopy using a labeled template/primer complex. By investigating the antibody effects on dimer formation of the heterodimeric enzyme, three domains corresponding to amino acids 230-300, 350-428, and residues around amino acid 540 involved in protein-protein interactions were localized.
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PMID:Structure-function relationships of HIV-1 reverse transcriptase determined using monoclonal antibodies. 137 37

During meiotic maturation and early embryonic cycles, the activity of maturation-promoting factor (MPF) cycles in exact correspondence with the mitotic cycles. For the appearance of MPF activity in starfish, protein synthesis is required except in the first meiotic cycle. In order to identify newly synthesized proteins involved in the regulation of MPF activity, we extracted poly(A)+ RNA from starfish eggs, and found that the egg poly(A)+ RNA induced germinal vesicle breakdown (GVBD) upon injection into immature oocytes of starfish and Xenopus. The molecular size of the poly(A)+ RNA responsible for GVBD was estimated to be approximately 22S by sucrose density gradient centrifugation. Since these characteristics of the starfish egg poly(A)+ RNA are similar to those of cyclin mRNAs from sea urchin and surf clam eggs, we synthesized a 50-mer antisense-cyclin oligonucleotide probe coding for a part of the sea urchin cyclin cDNA and used this to screen starfish RNA. The Northern blot analysis showed that the starfish egg RNA contained cyclin homologous transcripts. Incubation of the starfish egg poly(A)+ RNA and the antisense-cyclin oligonucleotide with RNase H completely destroyed its GVBD-inducing activity. These results indicated that starfish cyclin mRNA was the only poly(A)+ RNA responsible for GVBD. We constructed a starfish egg cDNA library to clone starfish cyclin cDNA. The longest cDNA clone containing 2190 base pairs was sequenced. The longest open reading frame consisted of 395 amino acid residues, and the predicted molecular size was 48 kDa. Comparison of the deduced amino acid sequences of starfish cyclin with known cyclins indicated that the starfish cyclin belongs to the B-type. Injection of synthetic mRNA of starfish cyclin caused GVBD in immature oocytes of starfish and Xenopus, while injection of synthetic mRNA of human CDC2 had no effect. The Northern blot analysis of starfish RNA extracted at various stages of the meiotic cycles suggested that the starfish cyclin transcript was stored in its polyadenylated form even in immature oocytes and was further polyadenylated at maturation.
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PMID:The starfish egg mRNA responsible for meiosis reinitiation encodes cyclin. 169 83

A phosphorothioate homocytidine 10-mer containing a cholesteryl moiety covalently linked to the 5'-end (Chol-SdC10) inhibited syncytium formation in susceptible T cells induced by HIV-1 and HIV-2. The syncytium inhibition effect was minimal with unmodified cytidine homopolymer of the same net charge. Chol-SdC10 was shown to protect CEM cells against infection by cell-free HIV-1 particles without any apparent toxicity to the growth of CD4+ T cells. The DNA polymerase activity of the purified reverse transcriptase (RT) of HIV-1 was markedly inhibited by Chol-SdC10 but the effect on the RNase H activity of RT was minimal. Analysis of the kinetics of reverse transcriptase inhibition mediated by the drug revealed that the inhibition at a higher concentration was competitive with respect to template primer binding and noncompetitive at lower concentrations. Chol-SdC10 also partially blocked the binding of gp120 to CD4 in a solid-phase ELISA. These results confirm that the anti-HIV activity of phosphorothioate cytidine homopolymers increases markedly by covalent modification with the cholesteryl moiety at the 5'-end and demonstrates that the cytoprotective effect is manifested at multiple steps in the virus life cycle. These steps include inhibition of retroviral replication activity as well as the binding and fusion of HIV with CD4+ T cells.
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PMID:Mode of action of 5'-linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncytia formation and infection by HIV-1 and HIV-2 in vitro. 170 17

alpha-Anomeric oligonucleotides are resistant to nucleases and display parallel hybridization with complementary DNA and RNA sequences. Although alpha-DNA/beta-RNA duplexes are not substrates for RNases H, alpha-oligos are able to inhibit translation through a RNase H independent mechanism. alpha-Oligos and their alpha-phosphorothioate analogs (12 mer) targeted against the splice acceptor site of the HIV-TAT gene were potent inhibitors of de novo HIV infection. Furthermore alpha-phosphorothioate and dithioate homo-oligomers exhibit an in vitro, nonsequence-specific, inhibitory effect on HIV reverse transcriptase.
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PMID:Alpha-oligonucleotides: a unique class of modified chimeric nucleic acids. 177 67

In order to inhibit the in vitro translation of Plasmodium falciparum mRNA coding for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS), oligodeoxynucleotides (ODNs) were directed against the translation initiation site or a site in the TS-coding region. In both cases considerable hybridization arrest, i.e. greater than 50% inhibition, was only achieved if the lengths of the ODNs to the two regions were 30 and 39 nucleotides, respectively, or longer. The ODN with the highest efficiency was a 49-mer directed against the TS-coding region (OTS49); 45 microM was sufficient to inhibit the expression of DHFR-TS by almost 90%. In this case the synthesis of DHFR-TS was interrupted at the binding site of OTS49 by a RNase H-independent mechanism. The resulting polypeptide was smaller (55 kDa) than one subunit of the native protein (71 kDa) and lacked TS activity.
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PMID:Hybridization arrest of cell-free translation of the malarial dihydrofolate reductase/thymidylate synthase mRNA by anti-sense oligodeoxyribonucleotides. 202 68


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