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)

The RNase H activity of recombinant HIV-1 reverse transcriptase (RT) has been characterized with respect to inhibition by azidothymidylate (AZTMP) and N-ethylmaleimide (NEM) and to cleavage patterns using either poly(rA)/poly(dT) or poly(rG)/poly(dC) as model substrate and either Mg2+ or Mn2+ as divalent cation activator. The inhibitory potency of AZTMP and other nucleotide analogues was found to be dependent on both the composition of the substrate and the divalent cation. The enzyme was significantly more sensitive to AZTMP inhibition with poly(rG)/poly(dC) than with poly(rA)/poly(dT) as substrate and in Mn2+ than in Mg2+ with either substrate. Kinetic studies indicated that AZTMP is a competitive inhibitor with respect to the substrate in Mn2+ whereas it behaves as an uncompetitive inhibitor in Mg2+. These results suggest that the enzyme may exist in two distinct forms depending on whether Mg2+ or Mn2+ is the divalent cation activator. Consistent with this suggestion is the alteration in the mode of cleavage of the substrate upon substitution of Mg2+ with Mn2+. In Mg2+, hydrolysis of poly(rA)/poly(dT) appears to be solely endonucleolytic, whereas in Mn2+, hydrolysis is both endonucleolytic and exonucleolytic. With poly(rG)/poly(dC) as substrate, hydrolysis is both endonucleolytic and exonucleolytic in either Mg2+ or Mn2+. There is a positive correlation between sensitivity to AZTMP and production of mononucleotides, suggesting that the exonuclease activity of RNase H is preferentially inhibited by AZTMP. The sensitivity of RNase H to inhibition by N-ethylmaleimide was also found to be markedly influenced by the substrate composition and the divalent cation activator, being most sensitive under conditions in which endonucleolytic activity predominates.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytically distinct conformations of the ribonuclease H of HIV-1 reverse transcriptase by substrate cleavage patterns and inhibition by azidothymidylate and N-ethylmaleimide. 750 46

Ribonuclease H (RNase H) recognizes a DNA-RNA hybrid duplex and catalyzes the hydrolysis of the phosphodiester linkages in only the RNA strand. Previously, we developed a method to cleave RNA in a sequence-dependent manner using RNase H and a complementary oligonucleotide containing 2'-O-methylribonucleosides. Since cleavage is restricted to a single site by the modified complementary strand, this system allows kinetic analysis of the RNase H reaction. We describe an investigation of the interactions between RNase HI from Escherichia coli and its substrate, and between the substrate and a metal ion using synthetic oligonucleotide duplexes modified at the cleavage site in combination with the 2'-O-methylribonucleotides. Firstly, the base moiety was changed to interfere with enzyme binding in either the major or minor groove. When 2-N-methylguanine was incorporated into the cleavage site, the Km value for this substrate, containing a methyl group in the minor groove, was 20-fold larger than that for the unmodified substrate, whereas 5-phenyluracil, with a phenyl group residing in the major groove of the duplex, did not affect the affinity. Secondly, the phosphodiester linkage at the cleavage site was changed into a phosphorothioate with a defined configuration. Only the Rp isomer was cleaved at this site in the presence of Mg2+ or Cd2+. These results suggest that the enzyme, but not the metal ion, interacts with the phosphate residue at the cleavage site. Thirdly, the 2'-position of the nucleoside on the 5'-side of the scissile phosphodiester was modified. Alteration of the 2'-hydroxyl function into an amino, fluoro or methoxy group, or removal of this 2'-hydroxyl group, did not affect the affinity for the enzyme, but reduced the reaction rate. An outer sphere interaction of a metal ion with the 2'-hydroxyl group is suggested.
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PMID:Studies of the interactions between Escherichia coli ribonuclease HI and its substrate. 752 71

The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.
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PMID:Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase. 754 83

A purified preparation of endogenous RNA-dependent DNA-polymerase (reverse transcriptase) earlier identified in rat brain (Ivanov, V.A., Pakhotin, P.I., Bobkova, N.V., and Ilyin, Yu.V. parallel Dokl. RAN (1992). V. 323, P. 173-177) has been obtained. A comparative analysis of the enzyme and recombinant reverse transcriptase coded by the mobile genome element jockey earlier expressed in a heterological cell system (Ivanov V.V., Melnikov A.A., Siunov A.V., Fodor I.I., Ilyin, Yu.V. parallel EMBO J. (1991), V. 10, P. 2489-2495) has been carried out. Like retroviral RNA-dependent DNA-polymerases, these enzymes show preference for polyribonucleotides and can use poly(rCm) as template. Besides they are inhibited by SH-reagents and require bivalent cations (Mg2+ or Mn2+) and detergent and/or KCl as ionic strength carrier. The enzymes differ drastically from retrovirus reverse transcriptases by a number of catalytic properties (low optima of concentration of requisite cations and ionic strength, strong preference for Mn2+, highly efficiency in using poly(rCm), lack of associated RNase H activity) but exhibit a high degree of similarity among themselves with regard to the above properties. It is suggested that endogenous reverse transcriptase from rat brain is a product of expression of the mobile genome element of the LINE family.
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PMID:[Endogenous cellular reverse transcriptase in the rat brain. A comparative analysis of the authentic enzyme and recombinant reverse transcriptase coded by a mobile genetic element of the LINE class]. 757 75

We have investigated the binding of human immunodeficiency virus reverse transcriptase (HIV-RT) to various hybrid RNA-DNA or DNA-DNA nucleic acid structures. Binding was measured by preequilibrating the RT with the nucleic acid substrate in the presence or absence of Mg2+ and then initiating synthesis or RNase H degradation reactions in the presence of excess "trap" polymer [poly(rA)-oligo(dT)]. The trap polymer sequestered RT molecules as soon as they dissociated from the substrate, such that the amount of synthesis or degradation on the substrate was proportional to the amount of bound RT. On hybrid substrates that had the 3' terminus of a complementary DNA oligomer recessed on a longer DNA or RNA template, binding to the RNA-DNA hybrid was more stable. Both the dissociation rate constant (k(off)) and equilibrium constant (Kd) values were larger for the DNA-DNA substrates by 5-10-fold. The difference was clearly in dissociation, since the association rate constant (k(on)) for both types of substrates was similar. On hybrid structures that had the 3' termini of a complementary RNA or DNA oligomer recessed on a longer DNA template, k(off) values are approximately the same on either structure. Although binding of the RT to DNA-DNA hybrid structures did not require Mg2+, its presence during the preequilibration period greatly stabilized binding. An approximate 20-60-fold decrease in the k(off), depending on the substrate structure, was observed with Mg2+. Measurements on one particular DNA-DNA hybrid indicated that the k(on) decreased by approximately 2 orders of magnitude with Mg2+. The relevance of these results to HIV replication is discussed.
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PMID:Parameters that influence the binding of human immunodeficiency virus reverse transcriptase to nucleic acid structures. 768 63

The recombinant 94 kDa Thermus thermophilus DNA polymerase (rTth pol) was found to release [33P]UMP when incubated with a RNA.DNA hybrid containing a [33P]UMP-labeled RNA strand. The RNase H activity was optimally active in the presence of low monovalent salt concentrations and when Mn2+ was used as the divalent cation activator. RNase H activity also was observed when Mg2+ replaced the Mn2+, but to a much lesser extent. A 60 nucleotide long, 5'- or 3'-radiolabeled RNA or DNA oligomer hybridized to a complementary DNA oligomer was used to determine the mode of digestion. The radiolabeled RNA.DNA hybrid or DNA.DNA duplex was incubated with rTth pol using various metal ion conditions and different incubation times. The DNA.DNA duplex showed very little enzymatic cleavage by rTth pol regardless of the Mn2+ or Mg2+ concentration. However, nearly complete digestion of the RNA.DNA hybrid was observed over a wide Mn2+ concentration range, thus demonstrating a preferential degradation of the RNA.DNA hybrid vs the DNA.DNA duplex. Time course reactions of the enzymatic digestion of the 3'-labeled RNA.DNA hybrid or DNA.DNA duplex by rTth pol indicated that digestion of the substrates occurred exonucleolytically in the 5'-->3' direction.
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PMID:Properties of the 5'-->3' exonuclease/ribonuclease H activity of Thermus thermophilus DNA polymerase. 771 Oct 21

A ribonuclease H activity from human placenta has been separated by ion exchange chromatography from the major RNase HI enzyme. Additional chromatographic steps allowed further purification, more than 3,000 fold compared to the crude extract in which it represents about 15% of the total RNase H activity. The enzyme requires Mg2+ ions for its activity, is strongly inhibited by the addition of Mn2+ ions or other divalent transition metal ions, and exhibits a pH optimum between 8.5 and 9. It shows a strong sensitivity to the SH-blocking agent N-ethylmaleimide. It has a strict specificity for double-stranded RNA-DNA duplexes and exhibits neither single-stranded nor double-stranded RNase (or DNase) activities. Therefore, this enzyme displays the characteristics of class II RNase H and is now termed RNase HII. Renaturation gel assays and gel filtration experiments proved a monomeric structure for the active enzyme with a native molecular weight of about 33 kDa. The human RNase HII acts as an endonuclease and releases oligoribonucleotides with 3'-OH and 5'-phosphate ends. It is therefore a candidate for the RNase H-mediated effect of antisense oligodeoxynucleotides.
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PMID:Purification and characterization of human ribonuclease HII. 781 13

To obtain more precise insight into the Mg(2+)-binding site essential for RNase HI catalytic activity, we have determined the crystal structure of E. coli RNase HI in complex with Mg2+. The analyzed cocrystal, which is not isomorphous with the Mg(2+)-free crystal previously refined at 1.48 A resolution, was grown at a high MgSO4 concentration more than 100 mM so that even weakly bound Mg2+ sites could be identified. The structure was solved by the molecular replacement method, using the Mg(2+)-free crystal structure as a search model, and was refined to give a final R-value of 0.190 for intensity data from 10 to 2.8 A, using the XPLOR and PROLSQ programs. The backbone structures are in their entirety very similar to each other between the Mg(2+)-bound and the metal-free crystals, except for minor regions in the enzyme interface with the DNA/RNA hybrid. The active center clearly revealed a single Mg2+ atom located at a position almost identical to that previously found by the soaking method. Although the two metal-ion mechanism had been suggested by another group (Yang, W., Hendrickson, W.A., Crouch, R.J., Satow, Y. Science 249:1398-1405, 1990) and partially supported by the crystallographic study of inactive HIV-1 RT RNase H fragment (Davies, J.F., II, Hostomska, Z., Hostomsky, Z., Jordan, S.R., Matthews, D. Science 252:88-95, 1991), the present result excludes the possibility that RNase HI requires two metal-binding sites for activity. In contrast to the features in the metal-free enzyme, the side chains of Asn-44 and Glu-48 are found to form coordinate bonds with Mg2+ in the metal-bound crystal.
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PMID:Crystal structure of Escherichia coli RNase HI in complex with Mg2+ at 2.8 A resolution: proof for a single Mg(2+)-binding site. 810 76

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.
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PMID:Metallobiochemistry of the magnesium ion. Characterization of the essential metal-binding site in Escherichia coli ribonuclease H. 830 92

In order to investigate the relationships between the three-dimensional structure and the enzymic activity of E. coli RNase HI, three mutant proteins, which were completely inactivated by the replacements of three functional residues, Asp10 by Asn (D10N), Glu48 by Gln (E48Q), and Asp70 by Asn (D70N), were crystallized. Their three-dimensional structures were determined by x-ray crystallography. Although the entire backbone structures of these mutants were not affected by the replacements, very localized conformational changes were observed around the Mg(2+)-binding site. The substitution of an amide group for a negatively charged carboxyl group in common induces the formation of new hydrogen bond networks, presumably due to the cancellation of repulsive forces between carboxyl side chains with negative charges. These conformational changes can account for the loss of the enzymic activity in the mutants, and suggest a possible role for Mg2+ in the hydrolysis. Since the 3 replaced acidic residues are completely conserved in sequences of reverse transcriptases from retroviruses, including human immunodeficiency virus, the concepts of the catalytic mechanism deduced from this structural analysis can also be applied to RNase H activity in reverse transcriptases.
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PMID:Crystal structures of ribonuclease HI active site mutants from Escherichia coli. 840 67


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