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At least 19 recordsLinked to original sources
PubMed1994

Metallobiochemistry of the magnesium ion. Characterization of the essential metal-binding site in Escherichia coli ribonuclease H.

Ribonuclease H (Escherichia coli) contains one strong magnesium-binding site, as determined by metal-titration experiments monitored by high field 1H-NMR and also by direct titration calorimetry. Kinetic and thermodynamic parameters were evaluated by 25Mg-NMR and were as follows: dissociation constant Kd, approximately 60 +/- 10 microM; activation free energy delta G*, approximately 49.8 +/- 0.9 kJ; on/off-rate for magnesium binding Kon, approximately 1.8 x 10(8) M-1 s-1, koff, approximately 1.1 x 10(4) s-1; quadrupole coupling constant chi B, 1.2 +/- 0.2 MHz. The dissociation constant was independently determined by standard analysis of 1H chemical shifts in magnesium-titration experiments and by microcalorimetry (Kd approximately 200 +/- 20 microM). Cobalt hexaamine, which also activates RNase H [Jou, R. & Cowan, J. A. (1991) J. Am. Chem. Soc. 113, 6685-6686], appears to bind at the same location as Mg2+(aqueous). Assignments of C2H and C4H protons to specific histidine residues have been made by two-dimensional correlated spectroscopy experiments. Direct 25Mg-NMR pH titrations show that an ionizable residue (pKa approximately 5.8), most likely one of the carboxylates in the active site, influences magnesium binding. On the basis of the magnesium coordination chemistry elucidated herein, recent proposals on active-site chemistry are critically assessed and general physicochemical aspects of magnesium-binding sites on proteins and enzymes are discussed.

Binding Sites↗
PubMed2001

Increased efficacy of antileishmanial antisense phosphorothioate oligonucleotides in Leishmania amazonensis overexpressing ribonuclease H.

Ribonuclease H (RNase H), an enzyme that cleaves an RNA sequence base-paired with a complementary DNA sequence, is proposed to be the mediator of antisense phosphorothioate oligonucleotide (S-oligo) lethality in a cell. To understand the role of RNase H in the killing of the parasitic protozoan Leishmania by antisense S-oligos, we expressed an episomal copy of the Trypanosoma brucei RNase H1 gene inside L. amazonensis promastigotes and amastigotes that constitutively express firefly luciferase. Our hypothesis was that S-oligo-directed degradation of target mRNA is facilitated in a cell that has higher RNase H activity. Increased inhibition of luciferase mRNA expression by anti-luciferase S-oligo and by anti-miniexon S-oligo in these stably transfected promastigotes overexpressing RNase H1 was correlated to the higher activity of RNase H in these cells. The efficiency of killing of the RNase H overexpressing amastigotes inside L. amazonensis-infected macrophages by anti-miniexon S-oligo was higher than in the control cells. Thus, RNase H appears to play an important role in the antisense S-oligo-mediated killing of Leishmania. Chemical modification of S-oligos that stimulate RNase H and/or co-treatment of cells with an activator of RNase H may be useful for developing an antisense approach against leishmaniasis. The transgenic Leishmania cells overexpressing RNase H should be a good model system for the antisense-mediated gene expression ablation studies in these parasites.

Animals↗
PubMed1994

Sequence specific cleavage of messenger RNA by a modified ribonuclease H.

Ribonuclease H (RNase H) is an endonuclease that cleaves only the RNA strand of an RNA-DNA hybrid to produce 5'-phosphate and 3'-hydroxy termini and lacks useful sequence specific recognition properties. A mutant form of the E. coli enzyme has been prepared that is suited for selective chemical modification at a site proximal to the substrate binding region. The chemical derivatization involves the formation of a disulfide linkage to a modified octadeoxyribonucleotide. The conjugate retains only 0.3% of the normal sequence independent RNase H activity demonstrating that substrate recognition can be modulated by a covalent appendage. A beta-globin RNA transcript containing a sequence complementary to that of the octadeoxyribonucleotide was cleaved in a catalytic fashion to two products upon treatment with the conjugate. The selectivity in the phosphodiester bond cleavage mediated by the conjugate was found to be different than that displayed by the nonderivatized enzyme. These results demonstrate the potential of semi-synthetic RNase H conjugates for mechanistic studies and their application as RNA targeted diagnostic or therapeutic agents.

Base Sequence↗
PubMed1990

Calf thymus ribonuclease H IIa activity lacks ribonuclease H specificity.

Less purified fractions of ribonuclease H IIa activity of calf thymus display divalent cation-dependent ribonuclease H activity and divalent cation-independent ribonuclease activity. Because the ratio of the two enzyme activities does not change during successive chromatographic procedures, we suggest that ribonuclease H IIa activity is indeed able to degrade both ssRNA and the RNA moiety of RNA.DNA-hybrids. Ribonuclease H IIa activity can therefore be differentiated from calf thymus ribonuclease H I and H IIb by its lack of ribonuclease H specificity. The native molecular mass of ribonuclease H IIa activity is between 23 and 28 kDa. Under denaturing conditions a 23 kDa-protein band copurifies with the enzyme activity suggesting that this enzyme is monomeric.

Animals↗
PubMed1999

Discrimination between ribonuclease H- and ribonuclease L-mediated RNA degradation by 2'-O-methylated 2-5A-antisense oligonucleotides.

2',5'-Oligoadenylate (2-5A) antisense chimeric oligonucleotides were synthesized containing varying 2'-O-methyl-ribonucleotide substitution patterns in the antisense domain. The ability of these composite oligonucleotides to mediate RNase H- and RNase L-catalyzed RNA degradation showed that these two enzymes have different activation requirements.

Adenine Nucleotides↗
PubMed1989

Serological analysis and characterization of calf thymus ribonuclease H IIb.

Ribonuclease H IIb, which seems to play a physiological role during transcription, was purified from calf thymus tissue. A polyclonal antibody, raised against the most purified ribonuclease H IIb fraction, recognizes in crude extracts almost exclusively a 52-kDa protein band. By immunoaffinity chromatography and immunoprecipitation experiments, we are able to deplete enzyme extracts from the crossreacting 52-kDa protein band and from ribonuclease H IIb activity. Enzyme activity is eluted from the immunoaffinity matrix in association with a 52-kDa protein under denaturing conditions. Immunoaffinity chromatography enables us also to calculate a purification factor of around 20,000 from the crude extract. The native molecular mass for the enzyme of around 45 kDa, as determined by gel filtration, suggests that calf thymus ribonuclease H IIb is most probably monomeric. The enzyme possesses an isoelectric point of 7.0. It requires Mg2+ ions for activity, is inhibited by N-ethylmaleimide, and exhibits a pH optimum of 9.0-9.5. The enzyme releases oligoribonucleotides with 3'-OH and 5'-phosphate ends, probably in an exonucleolytical manner. The third largest subunit of yeast RNA polymerase A (I) displays ribonuclease H activity [Huet et al. (1976) Nature 261, 431-433]. We discuss our findings in the light of a possible association of ribonuclease H IIb and RNA polymerase A (I) in higher eukaryotes.

Animals↗
PubMed1981

Nuclear location of ribonuclease H and increased level of magnesium-dependent ribonuclease H in rat liver on thioacetamide treatment.

Rat liver nuclei were isolated in aqueous solutions of low ionic strength or anhydrous glycerol. The presence of ribonuclease H (RNase H) [EC 3.1.4.34] activity in the cytoplasm is due to extraction of the nuclear enzyme by buffer and inorganic salts. Two forms of RNase H were separated from rat liver nuclei by affinity chromatography using a DNA-cellulose column. When the RNase H in the wash solution of nuclei with 0.3 M sucrose and in nuclear solution extracted with 0.15 M NaCl were fractionated on a single-stranded DNA-cellulose column, two peaks corresponding to Mn2+- and Mg2+-dependent RNases H were eluted at 0.1 M and 0.2 M NaCl, respectively, and a peak having both RNase H activities was recovered in the wash-through fraction from the column. Among the enzymes from these two fractions in the nuclei, the activity of the Mg2+-dependent RNase H which binds to DNA-cellulose increased several-fold within 24 h of a single injection of thioacetamide. The activities of Mg2+-dependent RNase H extracted with higher-salt solution from the nuclei and recovered in the flow-through fraction from the DNA-cellulose column and the Mn2+-dependent RNase H activities were relatively unaffected by an injection of thioacetamide.

Acetamides↗
PubMed1980

Purification, subunit structure, and serologicai analysis of calf thymus ribonuclease H I.

Calf thymus ribonuclease H I (for nomenclature, see Büsen, W., and Hausen, P. (1975) Eur. J. Biochem. 52, 179-190) has been purified to near homogeneity. The large scale purification procedure results in a 1,000-fold enrichment of enzyme protein over the crude extract. The enzyme has a molecular weight of about 80,000, and S value of about 5, and an isoelectric point of about 4.9 under nondenaturing conditions. The purified enzyme sample contains two forms of ribonuclease H I, possibly isozymes, named ribonuclease H I 1 and ribonuclease H I 2. They can be activated by Mn2+ or Mg2+ ions. The most highly purified fraction is composed of four polypeptides named A, B, C, and D with molecular weights of 31,6000, 26,6000, 24,800, and 24,300, respectively. Polypeptides A, C, and D are acidic, whereas Polypeptide B is basic. Each form consists of three polypeptides. Ribonuclease H I 1 and ribonuclease H I 2 have Polypeptides A and B in common and differ from each other in the third. The data are consistent with a trimeric (A, B, C/D) or tetrameric (A, B2, C/D) structure for calf thymus ribonuclease H I. When alkalisensitive supercoiled DNA molecules containing ribonucleotides covalently inserted in one of the DNA strands are used as substrate, the products of the reaction are relaxed circles; thus, ribonuclease H I has an endonucleolytic mode of action. The final preparation is free of ribonuclease, and also of endodeoxyribonuclease activity single- and double-stranded DNA. Rabbit antiserum raised against the most highly purified calf thymus ribonuclease H I specifically precipitates the Polypeptides A, B, C, and D and inhibits the Mn2+ - and Mg2+ -dependent enzyme activities to more than 90%. Whereas a typical monophasic neutralization curve is obtained with Mn2+ activation, the neutralization curve observed with Mg2+ is biphasic. These results and several other differences between the Mn2+ - and the Mg2+ -dependent activities of the ribonucleases H I seem best explained by a hypothesis in which the enzymes exist in two different conformations depending on the type of divalent cation activation. The antiserum neutralizes ribonuclease H I but not the other known calf thymus ribonuclease H activities (IIa; IIb), demonstrating that the different ribonuclease H activities in calf thymus are serologically distinct. Ribonuclease H I is localized in the cell nucleus as visualized by immunofluorescent staining of bovine cells.

Animals↗
PubMed1997

Importance of the C-terminal helix to the stability and enzymatic activity of Escherichia coli ribonuclease H.

The ribonuclease H (RNase H) family of enzymes selectively degrades the RNA strand of RNA-DNA hybrids. This activity is essential for retroviruses such as HIV and resides in a domain of the larger reverse transcriptase molecule. RNase H from Escherichia coli is the best-characterized member of the family and serves as a model for structure/function studies. Despite having almost identical alpha + beta folds, the isolated domain from HIV is inactive and much less stable than the E. coli homolog. The HIV domain also shows increased disorder in its C-terminal regions (E-helix and His-containing loop). We investigated the importance of this region by studying a variant of E. coli RNase H lacking these elements (RNHdeltaE). Despite the elimination of 33 of 155 residues (including a complete helix), this C-terminal deletion mutant folds cooperatively as a subdomain. Surprisingly, this protein lacking residues near the active site retains weak Mn2+-dependent activity. A peptide corresponding to the deleted E-helix is helical in isolation and stimulates the activity of the deletion mutant in vitro. These results have implications for the catalytic mechanism of RNase H and drug design targeted to HIV reverse transcriptase.

Amino Acid Sequence↗
PubMed1993

Reverse transcription of the Mauriceville plasmid of Neurospora. Lack of ribonuclease H activity associated with the reverse transcriptase and possible use of mitochondrial ribonuclease H.

The Mauriceville mitochondrial plasmid of Neurospora encodes a reverse transcriptase that synthesizes a full-length cDNA copy of the major plasmid transcript beginning directly opposite the 3' end of the template RNA (Kuiper, M. T. R., and Lambowitz, A. M. (1988) Cell 55, 693-704). Here, we show that the Mauriceville plasmid reverse transcriptase has no detectable RNase H activity and that cDNAs synthesized either by the column-purified reverse transcriptase or by the endogenous reverse transcriptase in purified ribonucleoprotein particles remain in a full-length duplex with the template RNA. The column-purified Mauriceville plasmid reverse transcriptase initiates cDNA synthesis by using short DNA primers, which remain attached to the 5' end of the (-) strand DNA (Wang, H., Kennell, J. C., Kuiper, M. T. R., Sabourin, J. R., Saldanha, R., and Lambowitz, A. M. (1992) Mol. Cell. Biol. 12, 5131-5144). We find that these primer DNAs can be precisely removed by S1 nuclease digestion of the initial cDNA.RNA duplex, suggesting a mechanism whereby this structure may contribute to primer removal in vivo. Finally, we show that Neurospora mitochondria contain an endogenous RNase H activity, which is present in mitochondrial ribonucleoprotein particle preparations prior to their purification. This mitochondrial RNase H can degrade the endogenous plasmid transcript in ribonucleoprotein particles in vitro and could play a similar role in vivo. The finding that the Mauriceville plasmid reverse transcriptase, which is believed to be a primitive enzyme, has no detectable RNase H activity is consistent with the hypothesis that retroviral reverse transcriptases acquired their RNase H domains from a gene encoding a cellular RNase H.

Amino Acid Sequence↗
PubMed1994

Characterization and subcellular localization of ribonuclease H activities from Xenopus laevis oocytes.

Ribonuclease H activities present in fully grown Xenopus oocytes were investigated by using either liquid assays or renaturation gel assays. Whereas the test in solution detected an apparently unique class I ribonuclease H activity, the activity gels did not detect this enzyme but another one with the molecular weight expected for a class II ribonuclease H. The ribonuclease HI was found to be primarily concentrated in the germinal vesicle, but around 5% of this activity was detectged in the cytoplasm and may correspond to the activity involved in antisense oligonucleotide-mediated destruction of messenger RNAs. The concentration of this class I ribonuclease H in oocytes is similar to that in somatic cells. The class II ribonuclease H remained undetectable by the test in solution because its activity was cryptic. On activity gel, a polypeptide with the apparent molecular mass of 32 kDa, expected for a ribonuclease HII, was found to be concentrated in mitochondria although no RNase H activity could be detected by using the liquid assay. Based on sedimentation studies, we hypothesize that the apparent absence of RNase H activity in solution could be the result of the association of this 32-kDa polypeptide with other polypeptides, or possibly nucleic acids, to form a multimer of, until now, unknown function.

Animals↗
PubMed1988

In addition to RNase H(70) two other proteins of Saccharomyces cerevisiae exhibit ribonuclease H activity.

Two ribonucleases H (RNases H) were purified to apparent homogeneity from the yeast Saccharomyces cerevisiae. The enzymes were separated from the previously described yeast ribonuclease H (RNase H(70), Karwan, R., Blutsch, H., and Wintersberger, U. (1983) Biochemistry 22, 5500-5507) by chromatography on Mono Q and blue-Sepharose columns and from each other on a Mono S column. The two proteins, RNase H(55) of molecular weight around 55,000 and RNase H(42) of molecular weight around 42,000, exhibit distinct enzymatic properties: RNase H(55) acts as a 5'-exonuclease of low specific activity and produces predominantly monoribonucleotides from the synthetic hybrid poly(rA)-poly(dT). RNase H(42) efficiently releases oligoribonucleotides from the same substrate. Polyclonal antibodies against these proteins do not cross-react with RNase H(70), and thus, these two RNases H probably do not represent proteolytic breakdown products of RNase H(70). Peptide maps obtained by total digestion of RNase H(55) and RNase H(42) with trypsin reveal several common peptides and, therefore, suggest that the two enzymes are related to each other. We tentatively conclude that RNase H(55) is proteolytically processed to RNase H(42) in vivo.

Amino Acids↗
PubMed1989

A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA-polymerase-alpha-primase complex.

A ribonuclease H which degrades RNA specifically in RNA-DNA hybrids and, moreover, stimulates its homologous DNA-polymerase-primase complex was purified from calf thymus. The enzyme consists of a single polypeptide of molecular mass 78 kDa. It requires divalent cations for activity, and prefers Mg2+ over Mn2+. Ribonuclease H is optimally active at neutral pH and in 75 mM potassium acetate and is strongly sensitive to N-ethylmaleimide. [3H]Poly(rA).poly(dT), [3H]poly(rC).poly(dI), and [3H]RNA.M13-DNA are degraded to 3-9-mer oligoribonucleotides with similar kinetics, whereas double- or single-stranded DNA, and double- and single-stranded RNA remain unaffected. The enzyme stimulates in vitro DNA synthesis by the immunoaffinity-purified calf-thymus DNA-polymerase-alpha-primase complex threefold. When ribonuclease H is present in a three-fold molar excess to the polymerase-primase complex, twice as much primer is formed as in the absence of ribonuclease H. Ribonuclease H also stimulates the elongation rate of DNA polymerase alpha by a factor of 2-3, independent of whether primase-primed DNA templates or templates primed with oligonucleotides are used. Our results suggest that this form of ribonuclease H is a likely candidate for a genuine primer-removing enzyme in mammalian cells.

Animals↗
PubMed1991

Class I and class II ribonuclease H activities in Crithidia fasciculata (Protozoa).

The protozoan Crithidia fasciculata contains two different ribonuclease H activities. These enzymes display similar physical and biochemical characteristics to their homologues in higher eukaryotes, for instance calf thymus class I and class II ribonuclease H. Class I ribonuclease H of lower and higher eukaryotes can be activated by Mg2(+)- and Mn2(+)-ions. However, the presence of Mn2(+)-ions is inhibitory for the Mg2(+)-dependent class II ribonuclease H activity of Crithidia fasciculata and calf thymus. The protozoan class I-homologue enzyme appears to be serologically related to the class I ribonuclease H of calf thymus.

Animals↗
PubMed1977

Ribonuclease H levels during the response of bovine lymphocytes to concanavalin A.

Several enzymes with ribonuclease H specificity have been identified in bovine lymphoid tissue. Their nomenclature and some of their properties have been reported previously. In this study the time course of the induction of the different ribonuclease H activities during the stimulation of resting bovine lymph-node cells with concanavalin A was investigated. The activity of one of these enzymes (ribonuclease H IIb) increases in parallel with the induction of uridine incorporation and is well separated from the induction of ribonuclease H I, which increases together with DNA synthesis. These data indicate that the different ribonuclease H activities serve different physiological functions. They suggest that ribonuclease H I belongs to the set of enzymes which are involved in DNA synthesis.

Animals↗
PubMed1974

Separation of ribonuclease H and RNA directed DNA polymerase (reverse transcriptase) of murine type-C RNA tumor viruses.

Ribonuclease H (RNA.DNA-hybrid ribonucleotidohydrolase, EC 3.1.4.34) has been reported to copurify with reverse transcriptase (RNA directed DNA polymerase) of RNA tumor viruses. In addition, viral specific ribonuclease H and reverse transcriptase of avian type-C viruses are thought to be part of the same polypeptide. In this report we show that a fraction of the ribonuclease H activity from Rauscher murine leukemia and Kirsten murine sarcoma viruses was separated from reverse transcriptase by anion exchange chromatography while the remaining portion co-purified with the viral polymerase. The amount of this co-purified nuclease activity was about 4- to 8-fold lower than the activity found in avian myeloblastosis virus (with respect to the ratio of ribonuclease H to reverse transcriptase) and this nuclease activity can only be detected by using labeled substrate of high specific radioactivity. However, a complete separation of ribonuclease H activity from reverse transcriptase was obtained by purifying core structures of the virus by sucrose density gradient centrifugation. While reverse transcriptase was present in the cores, there was no detectable ribonuclease H. Furthermore, a specific antibody against Rauscher leukemia virus reverse transcriptase did not inhibit any virion associated ribonuclease H activity. Our results suggest that in these virions these two enzyme activities reside in two separate molecules and probably in two different compartments of the virus. These findings emphasize a basic difference between the avian and murine type-C virus DNA polymerases.

Antibodies, Viral↗
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↗