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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two DNA strand transfer reactions occur during retroviral reverse transcription. The mechanism of the first, minus strand strong-stop DNA, transfer has been studied in vitro with human immunodeficiency virus 1 reverse transcriptase (HIV-1 RT) and a model template-primer system derived from the HIV-1 genome. The results reveal that HIV-1 RT alone can catalyze DNA strand transfer reactions. Two kinetically distinct ribonuclease (RNase) H activities associated with HIV-1 RT are required for removal of RNA fragments annealed to the nascent DNA strand. Examination of the binding of DNA.RNA duplex and single-stranded RNA to HIV-1 RT during strand transfer supports a model where the enzyme accommodates both the acceptor RNA template and the nascent DNA strand before the transfer event is completed. The polymerase activity incorporated additional bases beyond the 5' end of the RNA template, resulting in a base misincorporation upon DNA strand transfer. Such a process occurring in vivo during retroviral homologous recombination could contribute to the hypermutability of the HIV-1 genome.
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PMID:Mechanism of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. 127 6

The distantly related lentiviruses human immunodeficiency virus type 1 (HIV-1) and visna virus each encode a posttranscriptional regulatory protein, termed Rev, that is critical for expression of the viral structural proteins. We genetically mapped the cis-acting target sequence for visna virus Rev, the visna virus Rev-response element or RRE-V, to a complex 176-nucleotide RNA stem-loop structure that coincides with sequences encoding the N terminus of the transmembrane component of envelope. The computer-predicted structure of the RRE-V was validated by in vitro analysis of structure-specific RNase cleavage patterns. The visna virus Rev protein was shown to interact specifically with the genetically defined RRE-V in vitro but was unable to bind the HIV-1 RRE. Similarly, HIV-1 Rev was also unable to bind the RRE-V specifically. We therefore conclude that the HIV-1 and visna virus Rev proteins, while functionally analogous, nevertheless display distinct RNA sequence specificities. These findings provide a biochemical explanation for the observation that these two viral regulatory proteins are functional only in the homologous viral system.
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PMID:Structural and functional analysis of the visna virus Rev-response element. 131 70

Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA. This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities. In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme. The purified RT catalyzes a specific cleavage in HIV-1 RNA hybridized to tRNALys, the primer for HIV-1 reverse transcription. The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex. This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different. Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the HIV RNA-tRNALys complex. We refer to this unusual activity as RNase D. Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant HIV RT. The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column. Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity. Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNA-tRNALys at two distinct sites within the PBS sequence 5'-UGGCGCCCGA decreases ACAG decreases GGAC-3'. The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALys sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.
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PMID:Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase. 137 Oct 14

An in situ gel assay was applied to the study of double stranded RNA dependent RNase activity associated with reverse transcriptase (RT) of HIV-1 and murine leukemia virus. Polyacrylamide gels containing [32P] RNA/RNA substrate were used for electrophoresis of proteins under denaturing conditions. The proteins were renatured and in situ enzymatic degradation of 32P-RNA/RNA was followed. E. coli RNaseIII, but not E. coli RNaseH, was active in this in situ gel assay, indicating specificity of the assay to RNA/RNA dependent nucleases. Analysis of purified preparations of HIV-1 RT p66/p51 expressed in E. coli demonstrated an RNA/RNA dependent RNase activity comigrating with the large subunit (p66) of the enzyme. In addition, this activity of the RT was often accompanied by a contaminating RNA/RNA dependent RNase, with a molecular weight approximately 30,000 dalton identical to that of E. coli RNaseIII. As the p51 small subunit of HIV-1 RT and a mutant of RT p66/p51, at Glutamic acid #478, did not exhibit RNA/RNA dependent RNase activity, at least part of the active site of the RNA/RNA dependent RNase activity appeared to reside at the carboxy end of the molecule. As these RT proteins are also deficient of RNaseH, our results suggest overlapping or identical catalytic sites for degradation of the substrates RNA/DNA and RNA/RNA.
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PMID:Characterization of the double stranded RNA dependent RNase activity associated with recombinant reverse transcriptases. 138 38

Wheat DNA polymerase A has been purified from wheat germ. The previous purification procedure (Castroviejo, M. et al. (1979) Biochem. J. 181, 183-191; Tarrago-Litvak, L. et al. (1975) FEBS Lett. 59, 125-130), has been improved leading to a higher degree of purity. Several biochemical properties of the enzyme are described. Interestingly, wheat DNA polymerase A is able to copy natural poly(A)+ mRNA into cDNA, in a way that is similar to that of the human immunodeficiency virus reverse transcriptase (HIV-RT). All four dXTP and the oligo dT primer were required for cDNA synthesis. The cDNA product was completely digested in the presence of DNase I and predigestion of the mRNA template with RNase decreased dramatically the cDNA synthesis. The animal DNA polymerase gamma can not copy natural mRNA. Substances, known to alter the enzymatic activities have been used to compare enzymes properties. In the presence of glycerol, ethidium bromide or spermine, wheat DNA polymerase A, HIV-RT and DNA polymerase gamma behave similar and they differ from animal DNA polymerase alpha. Nevertheless, DNA polymerase A is more resistant than HIV-RT and DNA polymerase gamma to the chain terminator ddTTP, while the wheat enzyme is more inhibited than DNA polymerase gamma but more resistant than HIV-RT in the presence of N3-TTP.
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PMID:Wheat embryo DNA polymerase A reverse transcribes natural and synthetic RNA templates. Biochemical characterization and comparison with animal DNA polymerase gamma and retroviral reverse transcriptase. 169 Oct 20

The DNA polymerase and RNase H activities of HIV reverse transcriptase are both essential for HIV replication. Although the two activities are both catalyzed by a single polypeptide, they are physically separate; i.e., the DNA polymerase resides in the N-terminal domain whereas the RNase H is localized in the C-terminal domain. The present study was undertaken to characterize the enzymatic properties of these two activities and to determine whether the two catalytic sites are also functionally distinct. We have observed that EGTA specifically stimulates, whereas CaCl2 selectively inhibits, the RNA-dependent DNA polymerase activity but that neither compound has any effect on the RNase H activity of a recombinant HIV reverse transcriptase. The stimulation of the DNA polymerase activity by EGTA is dependent on the Mg2+ concentration; the greatest stimulation is observed at low Mg2+ concentrations. Similarly, the inhibition of DNA polymerase activity by Ca2+ is influenced by Mg2+ concentration. Ca2+ inhibition can be reversed by increasing Mg2+ concentrations, suggesting the possibility that CaCl2 inhibits the reverse transcriptase activity by competing for a metal-binding site on the enzyme. The pyrophosphate analogue phosphonoformate selectively inhibits the polymerase activity but not the RNase H activity of HIV reverse transcriptase. In contrast, the RNase H activity can be selectively inhibited by deoxyadenosine 5'-monophosphate, whereas the DNA polymerase activity is not inhibited. These results suggest that the DNA polymerase and RNase activities are not only physically separate but that they are also functionally distinct.
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PMID:Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase. 170 16

The crystal structure of the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) has been determined at a resolution of 2.4 A and refined to a crystallographic R factor of 0.20. The protein folds into a five-stranded mixed beta sheet flanked by an asymmetric distribution of four alpha helices. Two divalent metal cations bind in the active site surrounded by a cluster of four conserved acidic amino acid residues. The overall structure is similar in most respects to the RNase H from Escherichia coli. Structural features characteristic of the retroviral protein suggest how it may interface with the DNA polymerase domain of p66 in the mature RT heterodimer. These features also offer insights into why the isolated RNase H domain is catalytically inactive but when combined in vitro with the isolated p51 domain of RT RNase H activity can be reconstituted. Surprisingly, the peptide bond cleaved by HIV-1 protease near the polymerase-RNase H junction of p66 is completely inaccessible to solvent in the structure reported here. This suggests that the homodimeric p66-p66 precursor of mature RT is asymmetric with one of the two RNase H domains at least partially unfolded.
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PMID:Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. 184 17

HIV (human immunodeficiency virus) viraemia in serum or plasma of HIV-infected individuals was investigated by the polymerase chain reaction assay (PCR) in combination with reverse transcription to detect HIV-1 genomic RNA. Before PCR, plasma or serum was ultracentrifuged, precipitated virions were then treated with a RNase-free DNase, and a cDNA from the HIV-1 genomic RNA was synthesized. Thirty-three fresh plasma and seven sera from either HIV-1 antibody-positive individuals or patients treated with AZT were tested. Plasma from three patients were assayed 3 or 6 months apart. Twelve sera from HIV-1 antibody-negative individuals were used as negative control. PCR was performed with primers in LTR, gag and env regions: 11 of 40 samples were positive with three primer pairs, 16 with two primer pairs and 11 with only one primer pair. PCR on HIV-1 genomic cDNA was positive in 38 out of the 40 plasma or serum samples (95%), regardless of the clinical stage of the infection: HIV-1 was detected in 14 of the 15 untreated subjects and in 24 of the 25 AZT-treated patients. HIV p24 antigen was detected in the serum of 38% of subjects (15 of 40). The results suggest that this method is suitable for the detection of viral particles in plasma or serum from HIV-1-infected individuals irrespective of antiviral treatment.
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PMID:The polymerase chain reaction for the detection of HIV-1 genomic RNA in plasma from infected individuals. 171 16

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
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PMID:Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. 171 5

The RNA- and DNA-dependent DNA polymerase activities of two point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity have been compared to the wild-type enzyme activities using substrates consisting of an oligodeoxynucleotide primer hybridized to either a RNA or a DNA template. The RNase H phenotype had a negligible effect on the steady-state kinetics and processivity of reverse transcription of a homopolymer template-primer [poly(A).oligo(dT)]. However, analysis of the distribution of DNA products indicated that the ability of the mutants to reverse-transcribe a specifically primed 345-nucleotide heteropolymeric RNA template derived from the gag region of HIV-1 was impaired relative to the wild-type enzyme. Although the wild-type and mutant enzymes shared the same pause sites of synthesis along the RNA template, certain prematurely terminated nascent primer chains were poorly extended by the mutant enzymes and hence accumulated, suggesting that a catalytically functional RNase domain facilitated reinitiation of DNA synthesis at specific pause sites along a heteropolymer template. In contrast, the processivity and product distribution of DNA synthesis directed by a heteropolymer gag DNA template of the same nucleotide sequence were not significantly influenced by the RNase H phenotype of the mutants.
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PMID:Analysis of the RNA- and DNA-dependent DNA polymerase activities of point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity. 171 22


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