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)

Plus strand priming during retroviral reverse transcription requires specific cleavage within the polypurine tract of the viral genome by the reverse transcriptase-associated RNase H. Previously it has been shown that a 190-base RNA-DNA hybrid containing the Moloney murine leukemia virus polypurine tract can serve as a substrate for the priming reaction. To investigate the structural requirements for the reaction, a series of DNA oligonucleotides was hybridized to the 190-base single-stranded RNA and tested as substrates for RNase H. At low enzyme concentrations, the sites of cleavage are located 17-23 nucleotides from the 3'-end of the DNA oligonucleotide, consistent with the observations of others that binding of the DNA polymerase at a primer terminus fixes the position of cleavage by RNase H. At higher enzyme concentrations, additional cleavages are observed in the RNA 3' of these sites, but there is no preference for cleavage at the plus strand origin. In contrast to the results with DNA oligonucleotides, hybridization of RNA oligonucleotides containing the polypurine tract to the 190-base single-stranded DNA generates substrates that are cleaved at the origin and efficiently extended into DNA. An RNA oligonucleotide hybridized downstream of the polypurine tract is cleaved but not extended. These results support the view that RNase H cleavage to generate the plus strand primer is uncoupled from minus strand DNA synthesis.
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PMID:The use of DNA and RNA oligonucleotides in hybrid structures with longer polynucleotide chains to probe the structural requirements for moloney murine leukemia virus plus strand priming. 751 53

The accepted model of retroviral reverse transcription includes a circular DNA intermediate which requires strand displacement synthesis for linearization and creation of an integration-competent, long terminal repeat-flanked DNA product. We have used an in vitro model of this last step of reverse transcription to examine the role of the viral enzyme, reverse transcriptase (RT), in displacement synthesis. We show that Moloney murine leukemia virus RT possesses an activity which allows for displacement synthesis through a minimum of 1,334 bp of duplex DNA--an extent much greater than that required during in vivo reverse transcription and over 25-fold greater than has been previously demonstrated for a viral RT. RT does not function as a helicase in the classical sense but appears to closely couple duplex DNA melting with synthesis-driven translocation of the enzyme. In the absence of synthesis, the unwound region created by a primer-positioned RT appears to be no greater than 2 bp and does not advance along the template. Additionally, RT does not utilize ATP or any deoxynucleoside triphosphate not directly encoded by the template strand to catalyze processive duplex unwinding at a nick; nor does binding of the enzyme unwind duplex DNA in the absence of a 3' terminus. The approximate maximum chain elongation rate during strand displacement synthesis by Moloney murine leukemia virus RT falls between 0.73 and 1.5 nucleotides per s at 37 degrees C. The RNase H activity of RT does not appear to play a role in displacement synthesis; however, a 181-amino-acid C-terminal truncation of RT displays a dramatically reduced ability to catalyze synthesis through duplex DNA.
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PMID:Strand displacement synthesis capability of Moloney murine leukemia virus reverse transcriptase. 751 25

Specific, high-affinity RNA ligands to avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases were isolated from a combinatorial RNA library using the SELEX (systematic evolution of ligands by exponential enrichment) procedure. The selected RNA ligands bound their respective reverse transcriptases with approximately nanomolar dissociation constants. The ligands did not exhibit primary sequence conservation from selections against different target enzymes. Moreover, the selected ligands competed with the binding of template/primer complex and inhibited both the RNA-dependent DNA polymerase and the RNase H activities of the cognate reverse transcriptase. SELEX can yield both high-affinity and high-specificity oligonucleotide antagonists against specific members of a protein family.
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PMID:Selection of high-affinity RNA ligands to reverse transcriptase: inhibition of cDNA synthesis and RNase H activity. 751 91

Alpha-beta chimeric 17-mer oligodeoxyribonucleotides containing either 5, 10 or 15 beta nucleotides were synthesized. The stability of the RNA/chimera hybrids was only slightly affected by the alpha stretch and by the alpha-beta link, as was the affinity of the Moloney Murine Leukemia Virus reverse transcriptase for the duplexes. All chimeras inhibited in vitro cDNA synthesis in a cell-free system to various extent, via the degradation of the RNA target by RNase H.
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PMID:Chimeric alpha-beta oligonucleotides as antisense inhibitors of reverse transcription. 753 37

The RNase H domain of murine leukemia virus (MuLV) reverse transcriptase (RT) was replaced with Escherichia coli RNase H, and the effect on RNase H activity and processive DNA synthesis was studied, using RNA-DNA hybrids containing sequences from the MuLV polypurine tract (PPT). Two chimeric RTs, having the entire polymerase domain or all but the last 19 amino acids, were expressed. In both cases, these RTs made multiple cuts in PPT-containing substrates, whereas wild-type cleavages occurred primarily at sites consistent with the distance between the polymerase and RNase H active sites. Primer extension assays performed with the chimeric RTs, an RNase H-minus RT, and wild-type showed that the presence of a wild-type viral RNase H domain facilitates processive DNA synthesis. When wild-type RT was bound to primer-template, two retarded bands could be detected in band-shift assays. In the absence of primer extension, a high proportion of enzyme-bound primer-template was associated with the faster-migrating band, whereas with DNA synthesis, more of the bound radioactivity was in the super-shifted complex. This suggests that the super-shifted complex contains the active form of RT. The mutant RTs were deficient in formation of this complex, but the chimeric RTs were somewhat less defective than the RNase H-minus mutant. Our results demonstrate that in the wild-type enzyme, the RNase H domain is required to stabilize the interaction between RT and primer-template.
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PMID:Defects in primer-template binding, processive DNA synthesis, and RNase H activity associated with chimeric reverse transcriptases having the murine leukemia virus polymerase domain joined to Escherichia coli RNase H. 753 33

To determine the various roles of RNase H in reverse transcription, we generated a panel of mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase based on sequence alignments and the crystal structures of Escherichia coli and human immunodeficiency virus type 1 RNases H (S. W. Blain and S. P. Goff, J. Biol. Chem. 268:23585-23592, 1993). These mutations were introduced into a full-length provirus, and the resulting genomes were tested for infectivity by transient transfection assays or after generation of stable producer lines. Several of the mutant viruses replicated normally, some showed significant delays in infectivity, and others were noninfectious. Virions were collected, and the products of the endogenous reverse transcription reaction were examined to determine which steps might be affected by these mutations. Some mutants left their minus-strand strong-stop DNA in RNA-DNA hybrid form, in a manner similar to that of RNase H null mutants. Some mutants showed increased polymerase pausing. Others were impaired in first-strand translocation, independently of their wild-type ability to degrade genomic RNA, suggesting a new role for RNase H in strand transfer. DNA products synthesized in vivo by the wild-type and mutant viruses were also examined. Whereas wild-type virus did not accumulate detectable levels of minus-strand strong-stop DNA, several mutants were blocked in translocation and did accumulate this intermediate. These results suggest that in vivo wild-type virus normally translocates minus-strand strong-stop DNA efficiently.
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PMID:Effects on DNA synthesis and translocation caused by mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase. 753 10

The reverse transcriptase of retroviruses contains an RNase H activity essential for the proper synthesis of the viral DNA copy of the RNA genome. We have previously characterized a number of point mutations altering the RNase domain of the Moloney murine leukemia virus reverse transcriptase (S. W. Blain and S. P. Goff, J. Biol. Chem. 268:23585-23592, 1993). One such mutation, Y586F (a Y-to-F change at position 586), reduced RNase H activity, as assayed by in situ gel analysis, to about 5% of the wild-type level and prevented viral replication. We have now recovered a revertant virus with near-normal infectivity and in vitro enzymatic activity. The revertant contains a single substitution, N613H, distant in the primary sequence of the protein, but modeling with the Escherichia coli RNase H structure suggests that the reverted residue is close in space to the original substituted residue. Examination of the structure permits some suggestions as to how this second-site revertant restores enzyme activity.
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PMID:Reversion of a Moloney murine leukemia virus RNase H mutant at a second site restores enzyme function and infectivity. 754 47

Avian myeloblastosis virus and Maloney murine leukemia virus RNase H-reverse transcriptases pause when they encounter a 2'-5' linkage or a 2'-thiophosphate in their template RNAs, but eventually read through these backbone modifications. Both reverse transcriptases pause after the 2'-5' linkage but before the 2'-thiophosphate. These results suggest that in the absence of precise information concerning the behavior of a given reverse transcriptase with respect to a particular lesion or modification, caution should be exercised in the interpretation of primer extension data that is being used to determine the existence of, or map the position of, a crosslink, site of chemical modification or non-standard linkage in an RNA template.
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PMID:Reverse transcriptase reads through a 2'-5'linkage and a 2'-thiophosphate in a template. 754 85

Reverse transcription of a retroviral RNA genome requires two template jumps to generate the linear double-stranded DNA required for integration. The RNase H activity of reverse transcriptase has several roles during this process. We have examined RNase H cleavages that define the maximal 3' and 5' ends of Moloney murine leukemia virus minus strand DNA prior to the second template jump. In both the endogenous reaction and on model substrates in vitro, RNase H cleaves the genomic RNA template between the second and third ribonucleotides 5' of the U5/PBS junction, but other minor cleavages between 1 and 10 nucleotides 5' of this junction are also observed. Similar experiments examining the specificity of RNase H for tRNA primer removal revealed that cleavage generally leaves a ribo A residue at the 5' end of minus strand DNA. These observations suggest that three bases are typically duplicated on the ends of the minus strands, leading to an intermediate following the second jump which contains unpaired nucleotides. Model substrates mimicking the structure of this intermediate demonstrate that reverse transcriptase has little difficulty in utilizing such a branched structure for the initiation of displacement synthesis.
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PMID:Cleavage specificities of Moloney murine leukemia virus RNase H implicated in the second strand transfer during reverse transcription. 759 16

The active sites for the polymerase and nuclease activities of Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) reside in separate domains of a single polypeptide. We have studied the effects of RNase H domain mutations on DNA polymerase activity. These mutant RTs displayed decreased processivity of DNA synthesis. We also compared complexes formed between primer-templates and mutant and wild-type reverse transcriptase (RT). Although M-MuLV RT is monomeric in solution, two molecules of RT bound DNA cooperatively, suggesting that M-MuLV RT binds primer-template as a dimer. Some mutant RTs with decreased processivity failed to form the putative dimer.
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PMID:RNase H domain mutations affect the interaction between Moloney murine leukemia virus reverse transcriptase and its primer-template. 767 98


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