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)

Activities of the hepadnavirus polymerases are known to include those of DNA polymerase, reverse transcriptase and RNase H. To date, it has been difficult or impossible to clone and express the product as an active enzyme. In this study, full length capped RNA encoding Duck Hepatitis B Virus (DHBV) polymerase was produced by in vitro transcription from a T7 promoter. The RNA was translated in a rabbit reticulocyte lysate system and produced an 35S-Methionine labelled 79 Kd band on SDS-polyacrylamide gel electrophoresis. The translation product showed DNA polymerase and reverse transcriptase activities on exogenous templates (respectively) of DNA or RNA with random DNA hexamer primers. The same RNA transcripts were also microinjected into Xenopus oocytes, but appeared to be toxic and gave no detectable translation product. Production of hepadnavirus polymerase by in vitro transcription/translation may provide a useful tool for structure/function and pharmacological studies on this important group of polymerases.
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PMID:Duck hepatitis B virus polymerase produced by in vitro transcription and translation possesses DNA polymerase and reverse transcriptase activities. 128 90

The DNA replication system of bacteriophage T4 serves as a relatively simple model for the types of reactions and protein-protein interactions needed to carry out and coordinate the synthesis of the leading and lagging strands of a DNA replication fork. At least 10 phage-encoded proteins are required for this synthesis: T4 DNA polymerase, the genes 44/62 and 45 polymerase accessory proteins, gene 32 single-stranded DNA binding protein, the genes 61, 41, and 59 primase-helicase, RNase H, and DNA ligase. Assembly of the polymerase and the accessory proteins on the primed template is a stepwise process that requires ATP hydrolysis and is strongly stimulated by 32 protein. The 41 protein helicase is essential to unwind the duplex ahead of polymerase on the leading strand, and to interact with the 61 protein to synthesize the RNA primers that initiate each discontinuous fragment on the lagging strand. An interaction between the 44/62 and 45 polymerase accessory proteins and the primase-helicase is required for primer synthesis on 32 protein-covered DNA. Thus it is possible that the signal for the initiation of a new fragment by the primase-helicase is the release of the polymerase accessory proteins from the completed adjacent fragment.
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PMID:Protein-protein interactions at a DNA replication fork: bacteriophage T4 as a model. 131 Sep 46

DNA polymerase I (pol I) from Escherichia coli has three well-defined activities: DNA polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. We have raised monoclonal antibodies to pol I which selectively neutralize each of these three activities, thus supporting the model of separate active sites for each activity, heretofore exclusively demonstrated with proteolytic fragments of pol I. Antibodies from each class could bind pol I in the presence of antibodies of another class, indicating the existence of significant spatial separation between each of the three sites. In addition, several of the neutralizing antibodies were able to distinguish particular activities of the 5'-3' exonuclease. One of them, for example, inhibited the RNase H activity but not the DNase activity. Two other antibodies could, in addition to inhibiting the polymerase and the 3'-5' exonuclease, either stimulate or inhibit the 5'-3' exonuclease depending upon the assay conditions, particularly the ionic strength.
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PMID:Selective immunoneutralization of the multiple activities of Escherichia coli DNA polymerase I supports the model for separate active sites and indicates a complex 5' to 3' exonuclease. 132 12

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

We have studied a mutant Moloney murine leukemia virus with a deletion in reverse transcriptase (RT) which is predicted to make its RNase H domain resemble structurally that of human immunodeficiency virus RT. This deletion was based on improved RNase H homology alignments made possible by the recently solved three-dimensional structure for Escherichia coli RNase H. This mutant Moloney murine leukemia virus RT was fully active in the oligo(dT)-poly(rA) DNA polymerase assay and retained nearly all of wild-type RT's RNase H activity in an in situ RNase H gel assay. However, proviruses reconstructed to include this deletion were noninfectious. Minus-strand strong-stop DNA was made by the deletion mutant, but the amount of minus-strand translocation was intermediate to the very low level measured with RNase H-null virions and the high level seen with wild-type RT. The average length of translocated minus-strand DNA was shorter for the deletion mutant than for wild type, suggesting that mutations in the RNase H domain of RT also affect DNA polymerase activity.
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PMID:Defects in Moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H. 137 May 51

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

The reverse transcriptases (RTs) from human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively) are relatively highly related yet there are several significant differences in their catalytic activities. In an attempt to relate these functional dissimilarities to the differences in amino acid sequences, we have employed a novel approach of constructing chimeric molecules composed of complementary amino acid sequences derived from the two HIV RTs. These recombinant proteins were analyzed for their enzymatic activities and for their sensitivity to tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2[1H]-one and thione (TIBO), which selectively inhibits only HIV-1 RT. The active chimeric RTs were used to map the TIBO binding site on the HIV-1 RT molecules and to localize the putative sequences responsible for the high RNase H activity of HIV-1 RT relative to that of HIV-2 RT. The results suggest that TIBO interacts with amino acid residues located around residue 200 within the DNA polymerase domain of HIV-1 RT which shows a relatively low similarity to HIV-2 RT. The difference in the RNase H activity maps to a position in the DNA polymerase domain rather than to the RNase H domain. Out of the 12 chimeric RTs generated, four were either fully active or hyperactive, three others lost most of their catalytic activities, and the rest were totally inactive. The pattern of catalytic activities of these hybrid proteins can be explained by a model for the initial folding of HIV RTs, which entails the formation of three distinct and independently folded regions. Each region can be formed by amino acid sequences derived exclusively from either HIV-1 RT or HIV-2 RT.
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PMID:The catalytic functions of chimeric reverse transcriptases of human immunodeficiency viruses type 1 and type 2. 137 Dec 74

Luo and Taylor (J. Virol. 64:4321-4328, 1990) have previously shown that when, during RNA-directed DNA synthesis, a retroviral reverse transcriptase comes to a halt at the end of an RNA template, the associated RNase H produces a specific oligonucleotide that contains the 5' end of that template; in those studies the length of the oligonucleotide was predominantly 17 nucleotides. We have now investigated variables that might affect the formation and length of such a terminal oligonucleotide. We found small but significant variations in the length could be caused by the choice of reaction conditions and also the sources of reverse transcriptase and RNA template. Nevertheless, the general finding in all these situations was that RNase H acted at or about 14 to 18 nucleotides from the 5' end, thereby supporting the interpretation that in the reverse transcriptase, the cleavage site for the RNase H is held at around this distance behind the DNA polymerase activity. In other words, it appears that for the intact protein, the RNase H and reverse transcriptase activities may work in a coupled or coordinate manner. We also found that more than 80% of the residual 5' oligonucleotides remained base paired to the RNA-directed DNA product. Furthermore, under certain conditions, these short RNAs could act as efficient primers for an associated DNA-directed DNA synthesis in the reverse direction.
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PMID:When retroviral reverse transcriptases reach the end of their RNA templates. 137 69

A series of biochemical investigations to compare the DNA polymerase and RNase H functions of the reverse transcriptases (RTs) corresponding to azidothymidine (AZT)-sensitive and -resistant human immunodeficiency virus (HIV) strains are described. Steady-state kinetic studies with purified recombinant enzymes utilizing several templates and three inhibitors, 3' azido-3' deoxythymidine triphosphate (AZTTP), 3-amino-thymidine 5'-triphosphate, and 2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate, found consistent 2-4-fold differences between the enzymes from the two strains over a wide pH range. A strong pH dependence for all three inhibitors was found at pH values below 7.4 and suggested an ionizable group on the enzyme with a pK of about 7. The sensitivities of the RNase H activities of the two enzymes to AZTTP and AZTMP were also compared and found to be similar. The nucleotide incorporation fidelities of recombinant RTs corresponding to AZT-sensitive and -resistant clinical isolates were compared and the error specificities determined. No significant differences were found. Both enzymes were equally able to incorporate AZTTP into an elongating M13 DNA strand with concomitant chain termination. Purified wild-type and mutant virions from cell-culture supernatants were compared in "endogenous" DNA synthesis reactions, and the sensitivities of this activity to AZTTP were found to be similar. The contrast between the small differences found in this study and the high level of viral resistance in tissue culture presumably reflects an incomplete understanding of AZT inhibition of HIV in the cell.
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PMID:Biochemical studies on the reverse transcriptase and RNase H activities from human immunodeficiency virus strains resistant to 3'-azido-3'-deoxythymidine. 137 38

We have generated by site-directed mutagenesis plasmids that induce the synthesis of specific mutants of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). These recombinant mutants of HIV-1 RT, designed on the basis of our previous studies of HIV-1 and HIV-2 RTs, were analyzed for structure-function relationship by assessing their RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H activities. Three groups of mutants were studied. 1) We have investigated the importance of the only two sets of highly conserved double prolines found in the sequence of HIV-1 RT. The results indicate that the conversion of either one or both prolines (at positions 225 and 226) to threonines have no significant effect on all catalytic activities of the enzyme. The mutants in which prolines 419 and 420 were individually modified to threonines exhibit full activities, whereas the double proline 419/420 mutant lost most of its RNase H activity (although the DNA polymerase function was fully retained). 2) We have deleted phenylalanine 346 from HIV-1 RT, which is absent in wild type HIV-2 RT. This mutant of HIV-1 RT lost practically all catalytic activities. 3) A mutant of HIV-1 RT in which a cysteine residue substituted for alanine 446, was found to be slightly hyperactive for both DNA polymerase and RNase H activities.
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PMID:Functional analysis of novel selective mutants of the reverse transcriptase of human immunodeficiency virus type 1. 138 52


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