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)

Monoclonal antibodies were prepared against the avian myeloblastosis virus reverse transcriptase. These monoclonal antibodies specifically immunoprecipitated the alpha and beta subunits of the reverse transcriptase molecule, as well as the Pr180gag-pol precursor protein present in virus-infected cells. In addition, these monoclonal antibodies inhibited the DNA polymerase activity associated with the reverse transcriptase molecule but not the RNase H activity. The monoclonal antibody preparations were specific for the amino-terminal portion of the protein, as determined by the immunoprecipitation of a reverse transcriptase-beta-galactosidase fusion protein produced in Escherichia coli by molecular cloning procedures.
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PMID:Production and characterization of monoclonal antibodies against avian retrovirus reverse transcriptase. 618 37

The single-stranded DNA containing the Moloney murine leukemia virus origin for plus-strand synthesis was cloned in M13mp2 and used as a template for avian myeloblastosis virus reverse transcriptase in the presence of Moloney RNA which had been treated with pancreatic RNase A. The RNA pieces containing the polypurine stretch near the plus-strand origin were processed, presumably by RNase H, to generate primers for DNA synthesis which initiated both at the correct origin site and at one nucleotide downstream from the correct site. Approximately 50% of the labeled DNA fragments synthesized under these conditions retained the priming RNA on their 5' ends. When the isolated fragments were hybridized back to the template DNA and again treated with the reverse transcriptase, all of the RNA was removed from the labeled DNA. By using 5'-end-labeled pancreatic RNase A-resistant fragments, it was possible to show that the RNA primers were removed intact. It appears from these results that the RNase H activity associated with the enzyme shows a preference for cutting at the junction between the RNA and DNA moieties of such complexes and therefore is ideally suited for removing RNA primers.
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PMID:Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase. 619 10

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

We have determined the extent of RNA cleavage carried out during DNA synthesis by either human immunodeficiency virus (HIV) or avian myeloblastosis virus (AMV) reverse transcriptases (RTs). Conditions were chosen that allowed the analysis of the cleavage and synthesis performed by the RT during one binding event on a given template-primer. The maximum quantity of ribonuclease H (RNase H) sensitive template RNA left after synthesis by the RTs was determined by treatment with Escherichia coli RNase H. RNA cleavage products that were expected to be too short to remain hybridized, less than 13 nucleotides in length, were quantitated. Results showed that HIV- and AMV-RT degraded about 80% and less than 20%, respectively, of the potentially degradable RNA to these short products. Survival of longer, hybridized RNA was not a result of synthesis by a population of RTs that had selectively lost RNase H activity. Using an assay that evaluated the proportion of primers extended versus RNA templates cleaved during primer-extension by the RTs, we determined that essentially each molecule of HIV- and AMV-RT with polymerase also has RNase H activity. The results indicate that although both HIV- and AMV-RTs cleave the RNA template during synthesis, the number of cleavages per nucleotide addition with HIV-RT is much greater. They also suggest that some hybridized RNA segments remain right after the passage of the RT making the first DNA strand. In vivo, these segments would have to be cleaved or displaced in later reactions before second strand DNA synthesis could be completed.
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PMID:Quantitative analysis of RNA cleavage during RNA-directed DNA synthesis by human immunodeficiency and avian myeloblastosis virus reverse transcriptases. 752 28

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

RNA/DNA substrates derived from the 5' ends of human immunodeficiency virus (HIV) and Moloney murine leukemia virus (MMuLV) genomes were used to study the specificity of the RNase H activities of HIV, AMV (avian myeloblastosis virus), and MMuLV reverse transcriptases. These substrates were selected because they represent the site for the first template switch during proviral DNA synthesis. Variability of cleavage was observed depending on the origin of the enzyme as well as the sequence of the RNA/DNA substrate. The minimal size of hybrid recognized by the RNase H activity of reverse transcriptase was also affected by the same parameters, namely, the enzyme and the substrate origin. Moreover, the size of the residual 5'-undigested RNA after completion of the RNase H reaction depended on the position of the DNA annealed to the genomic RNA. When the hybrid was located at the 5' R region of the viral genome, stable hybrids with RNAs of 13-18 nucleotides remained following digestion by HIV reverse transcriptase, and 21-24 nucleotides following digestion by AMV reverse transcriptase and MMuLV reverse transcriptase. On the other hand, with all three enzymes, smaller sized hybrids remained when the DNA was hybridized to internal U5 or R sequences. The reason for this variance in size appears to be the inability of RNase H to efficiently digest at the 5' end of hybrid structures. Surprisingly, hybridization to the RNA template, of a DNA oligomer that extended 15 nucleotides beyond the 5' end of the RNA R region sequences, resulted in further digestion of the RNA. This unexpected mode of action of RNase H at the 5' end of the genomic RNA should be taken in consideration in studies of the first template switch.
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PMID:RNase H activity of reverse transcriptases on substrates derived from the 5' end of retroviral genome. 768 65

We have examined the ability of reverse transcriptases (RT) to catalyze strand transfer from internal regions of RNA templates, resulting in switching of a primer from one template to another. To study this phenomenon, we employed a system of donor and acceptor templates in which homologous strand transfer can occur from a homopolymeric sequence, positioned internally on the donor template. Our results indicate that reverse transcriptases from human immunodeficiency virus (HIV), avian myeloblastosis virus (AMV), and murine leukemia virus (MuLV) are all able to catalyze strand transfer from this sequence. Catalysis of this reaction is not dependent upon ribonuclease H (RNase H) activity, since an RNase H-deficient form of HIV-RT is able to catalyze the reaction efficiently. Additionally, N-ethylmaleimide, which inhibits RNase H but not polymerase activity, did not inhibit the template switching by either the native or RNase H-deficient forms of HIV-RT. Our data further indicate that template switching may be promoted by RT pausing at a specific site on the donor template. Conditions that increase RT pausing at this site also increase template switching. These results suggest that transient RT pausing at specific sites on the viral genome during reverse transcription may promote template switches that in turn lead to recombination.
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PMID:Pausing by retroviral DNA polymerases promotes strand transfer from internal regions of RNA donor templates to homopolymeric acceptor templates. 769 74

The 3SR (self-sustained sequence-replication) reaction is a very efficient method for isothermal amplification of target DNA or RNA sequences in vitro. This method requires three enzymatic activities: reverse transcriptase, DNA-dependent RNA polymerase and Escherichia coli ribonuclease H. We have modified the original protocol by using human immunodeficiency virus (HIV)-1 reverse transcriptase instead of avian myeloblastosis virus (AMV) reverse transcriptase to allow amplification with T7 RNA polymerase but without E. coli ribonuclease H. Comparison of the incorporation kinetics between the conventional three-enzyme 3SR and our two-enzyme 3SR shows differences in the kinetic behaviour. Furthermore, by the new two-enzyme 3SR, the amplified RNA is obtained in a purer form compared with the experiments with three-enzyme 3SR. The aim of our research is to adapt 3SR as a useful tool for darwinian evolutionary experiments.
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PMID:Comparison of self-sustained sequence-replication reaction systems. 863 38

We compared the thermal stabilities of wild-type recombinant avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) with those of mutants of the recombinant enzymes lacking RNase H activity. They differed in resistance to thermal inactivation at elevated temperatures in the presence of an RNA/DNA template-primer. RNase H-minus RTs retained the ability to efficiently synthesize cDNA at much higher temperatures. We show that the structure of the template-primer has a critical bearing on protection of RT from thermal inactivation. RT RNase H activity rapidly alters the structure of the template-primer to forms less tightly bound by RT and thus less able to protect the enzyme at elevated temperatures. We also found that when comparing wild-type or mutant AMV RT with the respective M-MLV RT, the avian enzymes retained more DNA synthetic activity at elevated temperatures than murine RTs. Enzyme, template-primer interaction again played the most significant role in producing these differences. AMV RT binds much tighter to template- primer and has a much greater tendency to remain bound during cDNA synthesis than M-MLV RT and therefore is better protected from heat inactivation.
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PMID:The role of template-primer in protection of reverse transcriptase from thermal inactivation. 1213 94

Ty1 reverse transcriptase/RNase H (RT/RH) is exquisitely sensitive to manganese concentrations. Elevated intracellular free Mn(2+) inhibits Ty1 retrotransposition and in vitro Ty1 RT-polymerizing activity. Furthermore, Mn(2+) inhibition is not limited to the Ty1 RT, as this ion similarly inhibits the activities of both avian myeloblastosis virus and human immunodeficiency virus type 1 RTs. To further characterize Mn(2+) inhibition, we generated RT/RH suppressor mutants capable of increased Ty1 transposition in pmr1 Delta cells. PMR1 codes for a P-type ATPase that regulates intracellular calcium and manganese ion homeostasis, and pmr1 mutants accumulate elevated intracellular manganese levels and display 100-fold less transposition than PMR1(+) cells. Mapping of these suppressor mutations revealed, surprisingly, that suppressor point mutations localize not to the RT itself but to the RH domain of the protein. Furthermore, Mn(2+) inhibition of in vitro RT activity is greatly reduced in all the suppressor mutants, whereas RH activity and cleavage specificity remain largely unchanged. These intriguing results reveal that the effect of these suppressor mutations is transmitted to the polymerase domain and suggest biochemical communication between these two domains during reverse transcription.
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PMID:Mn2+ suppressor mutations and biochemical communication between Ty1 reverse transcriptase and RNase H domains. 1753 63


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