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At least 19 recordsLinked to original sources
PubMed1975

Distinct ribonuclease H activities in calf thymus.

Three enzymes with ribonuclease H activity are present in calf thymus. They have been separated on the basis of chromatographic behaviour and molecular weight. They are further distinguished from one another by their ionic requirements and sensitivity to the -SH reagent N-ethylmaleimide. Two of these enzymes are classified as ribonuclease H, the third is obtained in a fraction which possesses ribonuclease H activity but also degrades double-stranded RNA and poly(rA). No association between any of the enzymes and cellular DNA polymerases could be demonstrated.

Ammonium Sulfate↗
PubMed1978

Ribonuclease H from rat liver. II. Partial purification and characterization of cytosol ribonuclease H1.

We have detected in rat liver cytosol three enzymes (termed C-1, C-2, and C-3) which cleaved the RNA moiety of RNA-DNA hybrid. These enzymes were separated from each other by DEAE-Sephadex and Sephadex G-200 chromatography. C-1 and C-2 specifically act on the RNA moiety of RNA-DNA hybrid, while C-3 degrades single-stranded RNA as well as the RNA of the hybrid. The molecular weights of C-1, C-2, and C-3 are about 110,000, 35,000 and 110,000 daltons, respectively, and their activities are absolutely dependent on divalent cations such as Mg2+ and Mn2+. Cleavage by C-1 and C-2 is endonucleolytic, producing mostly oligonucleotides and a small amount of mononucleotides which possess 3'-hydroxyl termini. It seems likely that C-2 is originally present in the nucleus and is released into cytosol because of its loose binding to the nuclear components. As for biochemical properties, C-1 is very similar to the cytosol ribonuclease H initially reported by Roewekamp and Sekeris, and C-2 is very similar to the nuclear ribonuclease H reported by us in the preceding paper.

Animals↗
PubMed1978

Ribonuclease H from rat liver. I. Partial purification and characterization of nuclear ribonuclease H1.

A ribonuclease H, an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrid, has been partially purified from rat liver nuclei and characterized. Neither native or denatured DNA, nor single or double-stranded synthetic polyribonucleotides were degraded by the enzyme. The enzyme possesses a molecular weight of about 36,000 and requires alkaline pH, magnesium ions, and ammonium sulphate for maximum activity. The enzyme acts on the hybrid as an endonuclease, resulting in oligonucleotides with 3'-hydroxyl termini. The properties of this enzyme were distinct from those of the rat liver cytosol enzyme reported by Roewekamp and Sekeris in many respects, such as molecular weight, optimal pH and requirements for divalent cations. Preliminary experiments suggest that the nuclear enzyme is localized in the nucleoplasm and nucleoli. These results indicate that multiple forms of ribonuclease H exist in different regions of rat liver cells.

Animals↗
PubMed1975

Adrenal Cortical Ribonuclease H (hybrid) (38494).

Ribonuclease Hybrid (RNaseH) from adrenal cortical tissue has been characterized. RNase H specifically degrades the RNA strand of purified RNA:DNA hybrids but is inactive on single- or double-stranded RNA or on DNA. The mode of clevage by RNase H is endonucleolytic, producing oligoribonucleoties with 3'-hydroxyl and 5'- phosphate termini. ACTH administration to guinea pigs results in no detectable change in adrenal cortical RNase H activity at times when changes in DNA synthesis are marked.

Adrenal Cortex↗
PubMed2025

Discovery of bimodal hepatitis B virus ribonuclease H and capsid assembly inhibitors.

Hepatitis B virus (HBV) ribonuclease H (RNaseH) inhibitors are a potent class of antivirals that prevent degradation of the viral pregenomic RNA during reverse transcription and block formation of mature HBV DNAs. Development of HBV RNaseH inhibitors is entering advanced preclinical analyses. To ensure the mechanism of action was fully understood, we defined the effects of RNaseH inhibitors on other steps of HBV replication. Some N-hydroxypyridinedione (HPD) HBV RNaseH inhibitors significantly reduced accumulation of capsids in HBV-replicating cells. A representative HPD 1466, with a 50% effective concentration against HBV replication of 0.25 µM, decreased capsid and core protein accumulation by 50-90% in HepDES19 and HepG2.2.15 cells. Surprisingly, 1466 did not affect pregenomic RNA encapsidation, demonstrating a specific effect on empty capsids. HBV genomic replication was not necessary for 1466's inhibitory effect as it decreased capsid accumulation in cells transfected with replication-deficient mutants blocking pgRNA encapsidation (Δ-bulge), DNA synthesis (YMHA), and RNaseH (D702A) activities. 1466 also decreased capsid and core protein accumulation in cells transfected with a core protein expression plasmid, indicating that other HBV products are unneeded. 1466 reduced initial capsid assembly rates in biochemical assembly reactions employing purified core protein (Cp149), demonstrating a specific effect on HBV core protein. We conclude that the bimodal HPD HBV RNaseH inhibitor 1466 is the prototypic member of a new class of capsid assembly modulators (CAM) that inhibits capsid assembly rather than accelerating it, as all other CAM classes do. We propose that this class be called CAM-I, for CAM-inhibitor. These results lay the foundation for identifying bimodal HBV antivirals targeting the RNaseH and capsid assembly.

Hepatitis B virus↗
PubMed1976

An assay of ribonuclease H, endoribonucleases, and phosphatases.

An assay for ribonuclease H (EC 3.1.4.34) is described which permits the estimation of the number and the type of cleavages produced by the enzyme when acting on the ribo moiety of a DNA-RNA hybrid substrate. With six different homopolymer hybrids tested, the number of enzymically induced breaks varied widely. The method is applicable to other endoribonucleases, phosphodiesterases, and phosphatases.

Endonucleases↗
PubMed1979

Two ribonucleases H from cultured plant cells.

Two forms of enzyme with ribonuclease H (RNase H) [EC 3.1.4.34] activities, have been partially purified from cultured plant cells, strain GD-2, derived from carrot root. One is an Mn2+-dependent RNase H, and the second is an Mg2+-dependent RNase H. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at around 0.2 M and 0.4 M potassium chloride in phosphocellulose chromatography, and were further purified using blue Sepharose. Mg2+-dependent RNase H exhibits maximal activity at pH 9.0, and requires 10 to 15 mM Mg2+ for maximal activity, whereas the Mn2+-dependent enzyme is most active at pH 8.0, is maximally active at an Mn2+ concentration of 0.4 mM, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. The enzymes liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. The apparent molecular weight of the Mg2+-dependent RNase H was estimated to 18--20 X 10(4) and that of the Mn 2+- dependent RNase H was estimated to be 14 x 10(4) by gel filtration.

Cells, Cultured↗
PubMed1978

Simplified method for purification of ribonuclease H from calf thymus.

An improved purification procedure for the isolation of ribonuclease H(hybrid nuclease; RNA-DNA-hybrid ribonucleotidohydrolase, EC 3.1.4.34) from the thymus of 4-to 6-months-old calves yields two highly active forms of the enzyme, designated as ribonuclease H1 and H2. Their isoelectric points are 5.0 +/- 0.05 and 5.25 +/- 0.05, respectively; their molecular weight, estimated from gel filtration, is in both cases 64,000 +/- 2000. On sodium dodecyl sulfate gel electrophoresis, two principal bands were identified, with molecular weights of 32,000 and 21,000. The nature of the nucleotides at the 3'-OH terminals, produced initially by the enzymic hydrolysis of hybridized RNA, was examined and shown to be a function of the divalent metal ion employed as activator.

Animals↗
PubMed1979

Multiple forms of ribonuclease H from rat liver cytosol.

Three forms (termed I, II, and III) of ribonuclease H (RNase H) [EC 3.1.4.34] activity are present in rat liver cytosol. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at 0 M, 0.25 M, and 0.5 M KCl in phosphocellulose chromatography, and were further purified by using blue Sepharose. They are further distinguished from one another by their ionic requirements, optimal pH, molecular weights, sedimentation coefficients, and sensitivity to the -SH reagent, p-chloromercuribenzoate, although I and III have similar characteristics. They liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini.

Animals↗
PubMed1979

Reverse transcriptase-associated ribonuclease H does not require zinc for catalysis.

omicron-Phenanthroline, a zinc chelating agent, is known to inhibit the DNA polymerase activity of cellular DNA-dependent and viral RNA-dependent DNA polymerases. We find that omicron-phenanthroline does not inhibit the reverse transcriptase-associated RNase H activity of retroviruses. Kinetic studies, using DNA template-primers as an inhibitor of RNase H, suggest that zinc does not play any role in template-primer binding by reverse transcriptase. These results also indicate a distinct binding site for the template and triphosphate substrates. Cellular RNase H from calf thymus and RNase H-II from Rauscher leukemia virus are likewise resistant to omicron-phenanthroline inhibition, implying non-involvement of zinc in the nucleic acid hydrolysis by these enzymes.

Avian Myeloblastosis Virus↗
PubMed1979

Identification of two different RNase H activities associated with yeast RNA polymerase A.

Two ribonuclease H activities have been found in yeast RNA polymerase A. The nuclease activities comigrated with subunits A49 (Mr = 49,000) and A40 (Mr = 40,000), after electrophoresis in a sodium dodecyl sulfate polyacrylamide gel containing [32P](rG)n . (dC)n as substrate. Both activities were also found, among other nucleases, in a high salt chromatin extract. Several lines of evidence suggest that the chromatin RNase H of 49,000 daltons (RNase H49) is the same protein as subunit A49. They co-migrate on sodium dodecyl sulfate-gel electrophoresis, have the same chromatographic properties, and dissociate simultaneously from RNA polymerase A. Fractions containing RNase H49 stimulate RNA synthesis by RNA polymerase A* lacking A49 and A34.5 subunits. Finally, limited proteolysis of the protein band having RNase H49 activity yields the characteristic fingerprint of the A49 subunit. This subunit, therefore, exists in two states: bound to chromatin and associated with RNA polymerase A. On the other hand, it is not yet clear whether the RNase H activity of 40,000 daltons, associated with RNA polymerase A, is due to the A40 subunit or whether it represents a trace contamination by a very active nuclease tightly bound to the enzyme.

Chromatin↗
PubMed1977

RNA-dependent DNA polymerase of avian sarcoma virus B77. I. Isolation and partial characterization of the alpha, beta2, and alphabeta forms of the enzyme.

Three forms of the RNA-dependent DNA polymerase were isolated from highly purified avian sarcoma virus B77 grown in duck embryo fibroblasts, using sequential chromatography on DEAE-cellulose, phosphocellulose, and poly(U)-cellulose. One form, which sedimented with about 5.2 S, contained only one species of polypeptide, with a molecular weight of 63,000; a second sedimented with about 7.8 S and contained only one species of polypeptide with a molecular weight of 81,000; and a third form, which sedimented with about 7.3 S, contained two species of polypeptides with molecular weights of 63,000 and 81,000. The molecular constitution of the three enzyme forms were therefore alpha, beta2, and alphabeta. All three possessed almost the same specific activity with poly(rA)-oligo(dT) as the primer-template. Forms alpha and alphabeta of avian sarcoma virus DNA polymerase have already been described in the literature; form beta2 is a new form. All three forms possessed ribonuclease H activity, the relative specific activities of the alpha, beta2, and alphabeta forms being about 1:4:5. All three enzyme forms were inhibited by antiserum to the alphabeta form, but whereas the alpha and alphabeta forms could be inhibited about 95%, the maximum degree of inhibition of the beta2 form was about 80%. The three enzyme forms also differed with respect to heat stability at 46 degrees, the monomeric alpha form of the enzyme being only about one-half as stable as the two dimeric forms.

Avian Sarcoma Viruses↗