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)

The avian retrovirus RNA-directed DNA polymerase contains an activity that is capable of removing hydrogen bonds from duplex nucleic acid molecules. This "unwinding-like" activity appears to be specific in its action, affecting RNA.DNA and DNA.DNA duplex molecules but not RNA.RNA duplexes. Studies with defined RNA.DNA hybrid molecules (e.g., Rous sarcoma virus RNA and complementary DNAs representing specific regions of the Rous sarcoma virus genome) and DNA.DNA duplexes indicate that, although this activity can remove a portion of the hydrogen bonds from these double-stranded structures, complete separation of complementary strands is not accomplished. The unwinding-like activity exhibits sensitivities to temperature and monovalent and divalent cation concentrations. It can also remove a specific large oligonucleotide from the 5' end of the viral genome subsequent to RNase H hydrolysis of viral RNA complexed to DNA present at that terminus. This reverse transcriptase-associated unwinding-like activity is discussed with respect to recently proposed models of retrovirus proviral DNA synthesis.
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PMID:Unwinding-like activity associated with avian retrovirus RNA-directed DNA polymerase. 7 11

Almost all DNA and RNA metabolizing enzymes can be assayed rapidly and very sensitively by exploiting the enhanced fluorescence of ethidium intercalated into duplex DNA or RNA. Denatured DNA and natural RNAs contain duplex regions due to intramolecular hydrogen-bonding and can also be sensitively measured. Where the product is truly single-stranded (e.g. dTn) it can be assayed by adding the appropriate complementary strand (e.g. dAn or rAn). Some of the assays described provide information not readily obtained by other assay procedures. Among the enzymes readily assayed are DNA and RNA polymerases, terminal deoxynucleotidyl transferases, nucleases of all varieties (e.g. single-strand specific, endonucleases including for example AP endonucleases, exonucleases, RNase H, etc.), ligases, topoisomerases including gyrases, and indirectly enzymes such as proteases and superoxide dismutase. DNA binding proteins such as histones and helix destablizing proteins can also be quantitatively assayed.
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PMID:Review: ethidium fluorescence assay. Part II. Enzymatic studies and DNA-protein interactions. 38 44

In the presence of Mg(2+) and a specific primer, ApG or GpG, the influenza WSN virion transcriptase synthesizes large, polyadenylic acid-containing complementary RNA (cRNA) (Plotch and Krug, J. Virol., 21:24-34, 1977). After removal of its polyadenylic acid with RNase H in the presence of polydeoxythymidylic acid, the in vitro cRNA distributed into seven discrete bands during electrophoresis in acrylamide gels containing 6 M urea. The eight known segments of virion RNA (vRNA) also distributed into seven bands under these conditions as two, rather than the expected three, large-sized segments were resolved. Each of the in vitro cRNA segments migrated slightly faster than the corresponding vRNA segment. To determine whether this difference in mobility reflects a difference in size between cRNA and vRNA, the double-stranded RNA formed by annealing labeled in vitro cRNA to unlabeled vRNA was subjected to various nuclease treatments and was analyzed by gel electrophoresis. Hybrids treated with RNase T2 or a combination of RNase T2 and RNase H migrated slightly faster than those treated only with RNase H, indicating that RNase T2 removed an RNA sequence other than polyadenylic acid, most probably a short sequence of vRNA not hydrogen bonded to cRNA. These results suggest that the in vitro cRNA segments are shorter than, and thus incomplete transcripts of the corresponding vRNA segments. All eight hybrids were resolved by gel electrophoresis, indicating that all eight vRNA segments are transcribed into cRNA in vitro. We also present evidence suggesting that the ApG primer initiates in vitro transcription exactly at the 3' end of vRNA.
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PMID:Segments of influenza virus complementary RNA synthesized in vitro. 62 84

The crystal structure of RNase H from Escherichia coli has been determined by the multiple isomorphous replacement method, and refined by the stereochemically restrained least-squares procedure to a crystallographic R-factor of 0.196 at 1.48 A resolution. In the final structure, the root-mean-square (r.m.s.) deviation for bond lengths is 0.017 A, and for angle distances 0.036 A. The structure is composed of a five-stranded beta-sheet and five alpha-helices, and reveals the details of hydrogen bonding, electrostatic and hydrophobic interactions between intra- and intermolecular residues. The refined structure allows an explanation of the particular interactions between the basic protrusion, consisting of helix alpha III and the following loop, and the remaining major domain. The beta-sheet, alpha II, alpha III and alpha IV form a central hydrophobic cleft that contains all six tryptophan residues, and presumably serves to fix the orientation of the basic protrusion. Two parallel adjacent helices, alpha I and alpha IV, are associated with a few triads of hydrophobic interactions, including many leucine residues, that are similar to the repeated leucine motif. The well-defined electron density map allows detailed discussion of amino acid residues likely to be involved in binding a DNA/RNA hybrid, and construction of a putative model of the enzyme complexed with a DNA/RNA hybrid oligomer. In this model, a protein region, from the Mg(2+)-binding site to the basic protrusion, covers roughly two turns of a DNA/RNA hybrid double helix. A segment (11-23) containing six glycine residues forms a long loop between the beta A and beta B strands. This loop, which protrudes into the solvent region, lies on the interface between the enzyme and a DNA/RNA hybrid in the model of the complex. The mean temperature factors of main-chain atoms show remarkably high values in helix alpha III that constitutes the basic protrusion, suggesting some correlation between its flexibility and the nucleic acid binding function. The Mg(2+)-binding site, surrounded by four invariant acidic residues, can now be described more precisely in conjunction with the catalytic activity. The arrangement of molecules within the crystal appears to be dominated by the cancelling out of a remarkably biased charge distribution on the molecular surface, which is derived in particular from the separation between the acidic Mg(2+)-binding site and the basic protrusion.
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PMID:Structural details of ribonuclease H from Escherichia coli as refined to an atomic resolution. 131 86

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.
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PMID:Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli. 164 58

The mechanism of RNase H substrate recognition is proposed from a model of a chemically modified DNA.RNA hybrid Escherichia coli RNase H complex. Site-directed mutagenesis of the enzyme and substrate titration observed by heteronuclear two-dimensional NMR spectra have been carried out. A model complex has been built, based on free structures of the enzyme and the substrate independently determined by x-ray crystallography and NMR distance geometry, respectively. In addition to steric and electrostatic complementarities between the molecular surfaces of the enzyme and the minor groove of the hybrid in the model, putative hydrogen bonds between the polar groups in the enzyme and 2'-oxygens of the RNA strand of the hybrid fix the hybrid close to the active site of the enzyme. The enzymatic activities of the mutant proteins and the changes in NMR spectra during the course of substrate titration are consistent with the present model. Moreover, the specific cleavage of the RNA strand in DNA.RNA hybrids can be explained as well as cleavage modes in modified heteroduplexes. A mechanism of enzymatic action is proposed.
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PMID:How does RNase H recognize a DNA.RNA hybrid? 166 98

Tertiary models of ribonuclease H (RNase H) domains in reverse transcriptases (RTs) from Moloney murine leukemia virus (MuLV) and human immunodeficiency virus (HIV-1) were built based upon the X-ray structure of RNase H from Escherichia coli (E. coli RNase H). In two models of RT-RNase H domains, not only active site residues but also residues, which construct a hydrophobic core and hydrogen bonds, are located in the same positions as those of E. coli RNase H. The whole backbone structure and the electrostatic molecular surface of MuLV RT-RNase H model are similar to those of E. coli RNase H. On the contrary, HIV-1 RT-RNase H model lacks the third helix and the following loop, resulting no positive charge clusters around the hybrid recognition site. Referring the complex models of RTs with their substrate hybrid, the interaction between DNA-polymerase and RNase H domains in RTs was discussed.
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PMID:Structural models of ribonuclease H domains in reverse transcriptases from retroviruses. 170 92

In vitro DNA synthesis on single-stranded circular DNA can be initiated by RNA primers. RNA chains are covalently extended by DNA polymerase II from KB cells and DNA polymerase I from Micrococcus luteus, but not by an RNA-dependent DNA polymerase from avian myeloblastosis virus. The reaction product consists of DNA chains with a piece of RNA at their 5'-ends, hydrogen bonded to the template DNA. The primer RNA is linked to the product DNA via a 3':5'-phosphodiester bond, and can be specifically removed by ribonuclease H. The possible role of ribonuclease H in RNA-primed DNA synthesis in vivo is discussed.
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PMID:RNA-primed DNA synthesis in vitro. 433 98

Reverse transcriptase isolated from avian myeloblastosis virus (AMV) and Rauscher murine leukemia virus (RLV) were examined for their ability to catalyze polymerization, ribonuclease H, pyrophosphate exchange, and pyrophosphorolysis reactions. A detailed characterization and a study of requirements for the expression of pyrophosphate exchange and pyrophosphorolysis reactions indicated that a variety of RNA and DNA template-primers supported these catalytic reactions. Furthermore, hydrogen bonding of template to primer was essential, although RNA:RNA template-primers, e.g. poly(rA) . (rU)9 or 70 S RNA . tRNA complex, were not utilized for these reactions. AMV enzyme required Mg2+, and RLV enzyme Mn2+, as the preferred divalent metal ion for the expression of these activities. Response of various catalytic reactions to site-specific inhibitors revealed that polymerization and pyrophosphate exchange reactions were susceptible to reagents that affected either the substrate or the template binding site, intrinsic zinc, or sulfhydryl groups. RNase H and pyrophosphorolysis activities, on the other hand, exhibited susceptibility only to the template site-specific reagent. We, therefore, conclude that RNase H and pyrophosphorolysis reactions are catalyzed through the template binding site while polymerization and pyrophosphate exchange reactions require additional participation of the substrate binding site, as well as that of intrinsic zinc and the presence of reactive sulfhydryl groups.
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PMID:Enzymatic activities associated with avian and murine retroviral DNA polymerases. Catalysis of and active site involvement in pyrophosphate exchange and pyrophosphorolysis reactions. 615 89

After thermal denaturation, an in vivo-labeled RNA was found in a temperature-sensitive initiation mutant of Bacillus subtilis (dna-37) associated with high-molecular-weight DNA. This RNA could be clearly distinguished from other RNA species by different techniques of separation, such as Sepharose 2B filtration, chromatography on nitrocellulose, and equilibrium centrifugation in density gradient. It was obtained even when HCHO was present during denaturation and chilling of nucleic acids and was still detected after a second denaturation as well as after incubation with proteinase K. Properties of the complex were not altered by prior treatment with RNase H. A control experiment using two samples of the complex treated either with pancreatic DNase or with pancreatic RNase, denatured together and centrifuged in the same density gradient, showed that no artifactual associations occur between the DNA and the RNA components of the complex. These results demonstrate that the DNA and RNA in the complex are associated by neither hydrogen bonds nor proteins, but are indicative of a DNA-RNA covalent linkage. In addition, during synchronous replication after a previous period at a nonpermissive temperature, DNA-linked RNA synthesis took place at specific times which coincided with the appearance of rifampin resistance of the first and the second replication cycles. A possible involvement of this RNA in the initiation of chromosome replication is discussed.
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PMID:Possible involvement of DNA-linked RNA in the initiation of Bacillus subtilis chromosome replication. 617 20


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