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)

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 handle region (residues 84-99) in ribonuclease HI (RNase HI) from Escherichia coli, which is rich in basic amino acid residues, was altered by alanine-scanning mutagenesis. Fifteen mutant proteins were purified to homogeneity and analyzed for the enzymatic activity. A mutation of either of 2 tryptophan residues at 85 or 90 resulted in a large increase in the Km value along with a large decrease in the Vmax value. These values probably resulted from conformational changes introduced by the mutations as indicated by the CD spectra of these mutant proteins. All other mutant enzymes had Vmax values similar to that of the wild-type enzyme. In contrast, replacement of any basic amino acid residue in the handle region, except for lysine 86, yielded proteins whose Km values were 3-5-fold higher than the wild-type enzyme. Such effects were shown to be cumulative, suggesting strongly that the cluster of positive charges in the handle region is important for the effective binding of the substrate. Interestingly, the region of human immunodeficiency virus reverse transcriptase with homology to E. coli RNase HI lacks the handle region which may account for the poor RNase H activity of the domain when separated from the polymerase domain.
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PMID:Importance of the positive charge cluster in Escherichia coli ribonuclease HI for the effective binding of the substrate. 164 12

Lys103 and Lys421 of Moloney murine leukemia virus reverse transcriptase have been implicated in the dNTP binding function as judged by their reactivity to a substrate binding site-directed reagent, pyridoxal 5'-phosphate (Basu, A., Nanduri, V. B., Gerard, G. F., and Modak, M. J. (1988) J. Biol. Chem. 263, 1648-1653). To assess the true catalytic importance of the individual lysine residues in Moloney murine leukemia virus reverse transcriptase, we mutated Lys103 and Lys421 to leucine and alanine, respectively. Analysis of the mutant enzymes revealed that mutation at the 103 position had a drastic effect on the DNA polymerase activity whereas the 421 mutation had no effect. Both mutants exhibited normal RNase H activity as well as the ability to bind to RNA or DNA templates as judged by UV-mediated cross-linking of the enzyme to the template primers. The enzyme with mutation at codon 421 (Lys----Ala) exhibited properties that were indistinguishable from the wild type with respect to its mode of catalysis, i.e. preference of template primer and divalent metal ion, RNA- or DNA-dependent DNA polymerase activity, RNase H activity, and the processive mode of DNA synthesis. These observations suggest that only Lys103 and not Lys421 is the catalytically important residue that is involved in the binding of substrate dNTP in Moloney murine leukemia virus reverse transcriptase.
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PMID:Site-directed mutagenesis of Moloney murine leukemia virus reverse transcriptase. Demonstration of lysine 103 in the nucleotide binding site. 169 72

Several groups have reported the use of antisense oligonucleotides to inhibit c-myc gene expression and study its biological role. However high concentrations of free oligonucleotides were generally needed. To lower their concentration and stabilize the antisense effect against c-myc, oligonucleotides were covalently linked to poly(L-lysine) and administered in ternary complexes formed with heparin (100 micrograms/ml). A sequence specific growth inhibition was observed at concentrations lower than 1 microM, while oligonucleotide-poly(L-lysine) conjugates alone were inefficient. Similar results occurred with other polyanionic compounds. Inhibition of proliferation was correlated to a reduction of c-myc protein and to a transient decrease in c-myc mRNA level. However, implication of RNase H in this process could not be demonstrated.
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PMID:Antiproliferative effects of antisense oligonucleotides directed to the RNA of c-myc oncogene. 170 28

The substrate deoxynucleoside triphosphate (dNTP) binding site of Moloney murine leukemia virus (M-MuLV) reverse transcriptase was labeled with pyridoxal 5'-phosphate (PLP), a substrate binding site-directed reagent for DNA polymerases (Modak, M. J. (1976) Biochemistry 15, 3620-3626). Treatment of M-MuLV reverse transcriptase with PLP results in the loss of RNA-dependent DNA polymerase activity, but has no effect on ribonuclease H activity. Neither template-primer nor substrate dNTP alone shows any protective effect from PLP-mediated inactivation. However, the presence of both template-primer and complementary substrate dNTP significantly protects M-MuLV reverse transcriptase from PLP inhibition. Using tritiated sodium borohydride to label the pyridoxylated enzyme, approximately 4 mol of PLP were incorporated per mol of enzyme. In the presence of template-primer and the complementary dNTP, however, only 2 mol of PLP were incorporated. Comparative tryptic peptide mapping of enzyme, modified in the presence and absence of substrates by PLP reaction on C-18 reverse phase columns, indicated the protection of two peptides from pyridoxylation in the presence of substrate triphosphate. These two peptides were further purified and characterized by amino acid analyses and sequencing and were found to span residues 103 to 110 and 412 to 425 in the primary amino acid sequence of M-MuLV reverse transcriptase. Furthermore, Lys-103 of peptide I and Lys-421 of peptide II were found to be the targets of pyridoxylation, indicating that these 2 lysine residues are involved in substrate dNTP binding in M-MuLV reverse transcriptase.
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PMID:Substrate binding domain of murine leukemia virus reverse transcriptase. Identification of lysine 103 and lysine 421 as binding site residues. 244 99

The nucleotide sequence of the human spumaretrovirus (HSRV) genome was determined. The 5' long terminal repeat region was analyzed by strong stop cDNA synthesis and S1 nuclease mapping. The length of the RU5 region was determined and found to be 346 nucleotides long. The 5' long terminal repeat is 1,123 base pairs long and is bound by an 18-base-pair primer-binding site complementary to the 3' end of mammalian lysine-1,2-specific tRNA. Open reading frames for gag and pol genes were identified. Surprisingly, the HSRV gag protein does not contain the cysteine motif of the nucleic acid-binding proteins found in and typical of all other retroviral gag proteins; instead the HSRV gag gene encodes a strongly basic protein reminiscent of those of hepatitis B virus and retrotransposons. The carboxy-terminal part of the HSRV gag gene products encodes a protease domain. The pol gene overlaps the gag gene and is postulated to be synthesized as a gag/pol precursor via translational frameshifting analogous to that of Rous sarcoma virus, with 7 nucleotides immediately upstream of the termination codons of gag conserved between the two viral genomes. The HSRV pol gene is 2,730 nucleotides long, and its deduced protein sequence is readily subdivided into three well-conserved domains, the reverse transcriptase, the RNase H, and the integrase. Although the degree of homology of the HSRV reverse transcriptase domain is highest to that of murine leukemia virus, the HSRV genomic organization is more similar to that of human and simian immunodeficiency viruses. The data justify classifying the spumaretroviruses as a third subfamily of Retroviridae.
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PMID:Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus. 245 55

Human immunodeficiency virus reverse transcriptase (HIV-RT) exhibits a strong sensitivity to pyridoxal 5'-phosphate (PLP), a substrate-binding site directed reagent for DNA polymerases (Modak, M. J. (1976) Biochemistry 15, 3620-3626). Treatment of HIV-RT with PLP followed by sodium borohydride reduction of the enzyme-PLP adduct results in irreversible inactivation of polymerase activity while RNase H activity associated with HIV-RT is minimally affected. Kinetic studies indicate that the PLP inhibition is complex. Yet one of the sites of PLP action appears to be involved in the process of dNTP binding as judged by (a) competitive mode of inhibition and (b) blockage of PLP into enzyme protein by the addition of substrate dNTP. Furthermore, this site is the only PLP reactive site which is accessible to borohydride reduction. Comparative tryptic peptide mapping of enzyme treated with PLP under a variety of conditions permitted the identification of a PLP reactive site containing peptide. Furthermore, reactivity of this site was also blocked by inclusion of substrate dNTP and appropriate template-primer. The amino acid composition and sequence analysis of this peptide showed that a lysine residue present at position 263 in the primary amino acid sequence of HIV-RT is the site of PLP reactivity. We therefore conclude that lysine 263 serves as an important part of the dNTP-binding domain in HIV-RT.
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PMID:Substrate binding in human immunodeficiency virus reverse transcriptase. An analysis of pyridoxal 5'-phosphate sensitivity and identification of lysine 263 in the substrate-binding domain. 247 Jul 47

A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus (HIV-1) reverse transcriptase (RT). The compounds can be grouped into two broad classes: nucleoside analogs and nonnucleoside inhibitors. The nonnucleoside inhibitors are quite specific for the polymerase activity of HIV-1 RT; they do not affect the polymerase activity of HIV-2 RT or the ribonuclease H (RNase H) activity of either HIV-1 RT or HIV-2 RT. Structural, biochemical, and genetic analyses showed that this group of inhibitors binds in a hydrophobic pocket near the polymerase active site. Mutations in amino acids that line this hydrophobic pocket, for example at tyrosine 181, tyrosine 188, or lysine 103, lead to enzymes that are resistant to the nonnucleoside inhibitors. We have investigated the enzymatic properties of two mutants of HIV-1 RT in which residues 181 and 188 were replaced by the corresponding amino acids in HIV-2 RT (tyrosine 181-->isoleucine and tyrosine 188-->leucine). The two tyrosine mutants closely resemble the wild-type HIV-1 RT in almost all the catalytic functions tested, including the heat stability, sensitivity of the DNA polymerase activity to inhibition by deoxynucleoside analogs, inhibition by the zinc chelator o-phenanthroline, and the Km values calculated for the DNA polymerase activity. There is, however, a slight difference in the effect of orthophenanthroline on the RNase H activity. In addition, there is a subtle disparity in the fidelity of DNA synthesis (analyzed by a mispair extension assay), thus indicating that these mutant RTs are not likely to confer any selective advantages or disadvantages to the variant virions over wild-type virus.
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PMID:Enzymatic properties of two mutants of reverse transcriptase of human immunodeficiency virus type 1 (tyrosine 181-->isoleucine and tyrosine 188-->leucine), resistant to nonnucleoside inhibitors. 752 32

We have investigated two regions of the viral RNA of human immunodeficiency virus type 1 (HIV-1) as potential targets for antisense oligonucleotides. An oligodeoxynucleotide targeted to the U5 region of the viral genome was shown to block the elongation of cDNA synthesized by HIV-1 reverse transcriptase in vitro. This arrest of reverse transcription was independent of the presence of RNase H activity associated with the reverse transcriptase enzyme. A second oligodeoxynucleotide targeted to a site adjacent to the primer binding site inhibited reverse transcription in an RNase H-dependent manner. These two oligonucleotides were covalently linked to a poly(L-lysine) carrier and tested for their ability to inhibit HIV-1 infection in cell cultures. Both oligonucleotides inhibited virus production in a sequence- and dose-dependent manner. PCR analysis showed that they inhibited proviral DNA synthesis in infected cells. In contrast, an antisense oligonucleotide targeted to the tat sequence did not inhibit proviral DNA synthesis but inhibited viral production at a later step of virus development. These experiments show that antisense oligonucleotides targeted to two regions of HIV-1 viral RNA can inhibit the first step of viral infection--i.e., reverse transcription--and prevent the synthesis of proviral DNA in cell cultures.
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PMID:Sequence-specific inhibition of human immunodeficiency virus (HIV) reverse transcription by antisense oligonucleotides: comparative study in cell-free assays and in HIV-infected cells. 756 37

Site-directed mutagenesis has been used to assess the importance of lysine 263 in substrate binding of human immunodeficiency virus-1 (HIV-1) reverse transcriptase. Previous studies have indicated that lysine 263 functions in the binding of 2'-deoxynucleoside 5'-triphosphate (dNTP) substrates (Basu, A., Tirumalai, R. S., and Modak, M. J. (1989) J. Biol. Chem. 264, 8746-8752). We studied this interaction directly by using site-specific mutagenesis to change lysine 263 to a serine. Highly purified mutant enzyme K263S bound natural dNTP substrates and primed polynucleic acid substrates with equal affinity when compared to the wild type reverse transcriptase. No difference was observed in the binding of 3'-azido-2',3'-dideoxythymidine 5'-triphosphate to the mutant reverse transcriptase on the basis of Km and Ki determinations. The serine substitution had no effect on RNase H activity. These results indicate that lysine 263 is not essential in the binding of substrates to HIV-1 reverse transcriptase.
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PMID:Biochemical analysis of human immunodeficiency virus-1 reverse transcriptase containing a mutation at position lysine 263. 767 98


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