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)

We have previously described the in vitro and in vivo characterization of a panel of mutations affecting the RNase H domain of Moloney murine leukemia virus reverse transcriptase (Blain, S. W., and Goff, S.P. (1993) J. Biol. Chem. 268, 23585-23592; Blain, S. W., and Goff, S. P. (1995) J. Virol. 69, 4440-4452). We were intrigued by a discrepancy between in vitro and in vivo RNase H results for two of the mutants. While delta C and delta 5E appeared to have nearly wild-type RNase H activity in vitro, they were unable to degrade their genomic RNA in vivo and thus were effectively RNase H null mutants in this context. In this present report, we describe the differential effects of these mutations on RNase H activity in vitro in the presence of Mg2+ versus Mn2+: mutants delta C and delta 5E were active in the presence of the less biologically relevant Mn2+ and not in the presence of Mg2+. We also describe three mutants with only partial activity in Mg2+. The presence of the different cations can also affect DNA polymerization and processivity of an RNase H-deficient mutant.
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PMID:Differential effects of Moloney murine leukemia virus reverse transcriptase mutations on RNase H activity in Mg2+ and Mn2+. 857 37

Recently we demonstrated that the p58 subunit of p68/p58 HIV-2 reverse transcriptase (RT) heterodimer, produced by processing of p68/p68 homodimer with recombinant HIV-2 protease, terminates at Met484 [Fan, N., et al. (1995) J. Biol. Chem. 270, 13573-13579]. Here we describe purification and characterization of the p68/p58 heterodimer of recombinant HIV-2 RT. It exhibited both RT and RNase H activities, obeyed Michaelis-Menten kinetics, and was competitively inhibited by the DNA chain terminator ddTTP (Ki[app] = 305 +/- 20 nM). The HIV-2 RT-associated RNase H exhibited a marked preference for RNA hydrolysis from a HIV-1 gag-based heteropolymeric RNA/DNA hybrid in the presence of either Mg2+ or Mn2+, compared to the [3H]poly(rA).poly(dT) or [3H]poly(rG).poly(dC) homopolymeric substrates. Relative to HIV-1 RT, the RNase H activity of HIV-2 RT was only 5% toward the [3H]poly(rA).poly(dT) in the presence of Mg2+. The size distribution of products generated from [3H]poly(rA).poly(dT) by HIV-2 RT-associated RNase H was markedly distinct from that of HIV-1 RT in the presence of Mg2+ or Mn2+. The p68/p58 HIV-2 RT heterodimer, produced by specific cleavage using HIV-2 protease, should be useful for inhibition and biophysical studies aimed at discovering and designing drugs directed toward HIV-2.
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PMID:Characterization of the p68/p58 heterodimer of human immunodeficiency virus type 2 reverse transcriptase. 863 74

Bacteriophage T4 RNase H is a 5' to 3' exonuclease that removes RNA primers from the lagging strand of the DNA replication fork and is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases. The crystal structure of the full-length native form of T4 RNase H has been solved at 2.06 angstroms resolution in the presence of Mg2+ but in the absence of nucleic acids. The most conserved residues are clustered together in a large cleft with two Mg2+ in the proposed active site. This structure suggests the way in which the widely separated conserved regions in the larger nucleotide excision repair proteins, such as human XPG, could assemble into a structure like that of the smaller replication nucleases.
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PMID:Structure of bacteriophage T4 RNase H, a 5' to 3' RNA-DNA and DNA-DNA exonuclease with sequence similarity to the RAD2 family of eukaryotic proteins. 867 16

RNA hybridized to 2'-O-methyloligoribonucleotides and incubated in nuclear extracts from HeLa cells is truncated, resulting in a distinct product terminated at the 5' end of the antisense oligonucleotide. The activity responsible for this effect is not RNase H but rather a novel exonuclease degrading RNA in the 3' to 5' direction. The enzymes requires ATP and Mg2+ ions. Except for dATP, no other nucleoside triphosphate or nonhydrolyzable ATP analog supports the exonucleolytic activity. In spite of the nuclear origin and activity requirements similar to those required for pre-mRNA splicing, the exonuclease operates with equal efficiency on intron-containing and intronless RNAs, excluding the possibility that it is associated with the splicing machinery.
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PMID:Antisense 2'-O-methyloligoribonucleotides hybridized to RNA block a nuclear, ATP-dependent 3'-5' exonuclease. 878 94

The reverse transcriptase-associated RNase H activity of Moloney murine leukemia virus specifically cleaves within the polypurine tract region of the viral genome to generate the primer for plus-strand DNA synthesis and removes the tRNA primer after minus-strand initiation by preferentially cleaving the RNA one nucleotide before the RNA-DNA junction. Moreover, the enzyme is unable to cleave the extended tRNA substrate at the RNA-DNA junction even at high enzyme concentrations. The RNase H domain of the reverse transcriptase was expressed as a glutathione S-transferase fusion protein and purified from Escherichia coli extracts. Following removal of the glutathione S-transferase portion of the protein, the specificity of the isolated RNase H domain was determined in the plus-strand primer reaction and in the tRNA primer removal reaction. Although the isolated domain lacked specificity in both cases, it was still unable to cleave the tRNA substrate precisely at the RNA-DNA junction. Specificity in both cases could be restored by adding back a truncated form of Moloney murine leukemia virus reverse transcriptase lacking the RNase H domain. These results implicate the polymerase domain as a specificity determinant for the RNase H activity of reverse transcriptase. The isolated RNase H domain had higher activity in the presence of Mn2+ than in the presence of Mg2+, but neither the RNase H domain alone nor the RNase H domain coupled to the polymerase domain in wild-type protein exhibited the normal cleavage specificities in the presence of the nonphysiological divalent cation.
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PMID:RNase H domain of Moloney murine leukemia virus reverse transcriptase retains activity but requires the polymerase domain for specificity. 897 Sep 88

A recently introduced thermostable RNase H was tested to determine its effectiveness in RNase H mapping reactions. Procedures are described which should have general use with both the thermostable and the Escherichia coli RNase H enzymes. Using the thermostable RNase H at higher temperatures extends the range of oligodeoxyribonucleotide/RNA combinations that yield satisfactory results. Northern blot analyses of total RNA was used to demonstrate that native RNAs can be analyzed by oligodeoxyribonucleotide directed RNase H digestion with minimal sample processing as long as care is taken to maintain thermal stringency both during reaction assembly and termination. Increased thermal stringency allows for higher DNA concentrations to ensure complete site-specific digestion of target RNAs or to permit simultaneous cleavage with multiple oligodeoxyribonucleotides. Partial digests can also be controlled by manipulating oligodeoxyribonucleotide concentrations. In addition, the thermostable RNase H was shown to be active at magnesium ion concentrations as low as 0.1 mM. This allows for optimization of Mg2+ effects on overall sample integrity and DNA/RNA interactions over at least a 20-fold range (2.0-0.1 mM).
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PMID:Use of thermostable and Escherichia coli RNase H in RNA mapping studies. 917 89

Bacteriophage T4 RNase H, which removes the RNA primers that initiate lagging strand fragments, has a 5'- to 3'-exonuclease activity on DNA.DNA and RNA.DNA duplexes and an endonuclease activity on flap or forked DNA structures (Bhagwat, M., Hobbs, L. J., and Nossal, N. J. (1997) J. Biol. Chem. 272, 28523-28530). It is a member of the RAD2 family of prokaryotic and eukaryotic replication and repair nucleases. The crystal structure of T4 RNase H, in the absence of DNA, shows two Mg2+ ions coordinated to the amino acids highly conserved in this family. It also shows a disordered region proposed to be involved in DNA binding (Mueser, T. C., Nossal, N. G., and Hyde, C. C. Cell (1996) 85, 1101-1112). To identify the amino acids essential for catalysis and DNA binding, we have constructed and characterized three kinds of T4 RNase H mutant proteins based on the possible roles of the amino acid residues: mutants of acidic residues coordinated to each of the two Mg2+ ions (Mg2+-1: D19N, D71N, D132N, and D155N; and Mg2+-2: D157N and D200N); mutants of conserved basic residues in or near the disordered region (K87A and R90A); and mutants of residues with hydroxyl side chains involved in the hydrogen bonding network (Y86F and S153A). Our studies show that Mg2+-1 and the residues surrounding it are important for catalysis and that Lys87 is necessary for DNA binding.
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PMID:Identification of residues of T4 RNase H required for catalysis and DNA binding. 935 15

We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the -1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMTV RT were compared with those of the well-studied RT of HIV-1, the causative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivity to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs), as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV-1 RT.
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PMID:Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization. 944 85

We have cloned, expressed, and purified to electrophoretic homogeneity a human RNase H. The enzyme has a molecular weight of 32 kDa, is Mg2+ dependent, and is inhibited by Mn2+ and N-ethylmaleimide. Its molecular weight and cleavage characteristics are consistent with type 2 human RNase H. The human RNase H we have cloned is highly homologous to Escherichia coli RNase HI (33.6% amino acid identity) and to other RNase H enzymes homologous to E. coli RNase HI. The enzyme is encoded by a single gene that is at least 10 kb in length and is expressed ubiquitously in human cells and tissues.
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PMID:Molecular cloning and expression of cDNA for human RNase H. 951 96

Reverse transcriptase (RT)-associated ribonuclease H (RNase H) can cleave both the RNA template of DNA/RNA hybrids as well as double-stranded (ds) RNA. This report shows that human immunodeficiency virus (HIV)-RT can also cleave the template strand of dsDNA when Mg2+ is replaced by Fe2+ in the RNase H active site of HIV-RT. The cleavage mechanisms as well as the positions of the cut vary depending on whether RNA or DNA is used. While DNA is cleaved 17 base positions upstream of the primer 3'-end, RNA is cleaved 18 base positions upstream. Competition experiments show that Fe2+ replaces the catalytically active Mg2+ of RT-associated RNase H. The bound Fe2+ is the source of locally generated OH-radicals that cleave the most proximate base in the DNA. Electrophoretic mobility studies of the cleaved fragments suggest that DNA is cleaved by an oxidative mechanism, while RNA is cleaved by an enzymatic mechanism which is indistinguishable from the Mg2+-dependent cleavage. The Fe2+-dependent cuts can be used to trace the active site of RT-associated RNase H on dsDNA as well as on dsRNA and DNA/RNA hybrids. The observed 1 base difference in the cleavage positions on DNA and RNA templates can be attributed to conformational differences of the bound nucleic acids. We suggest that the lower pitch of dsRNA and DNA/RNA hybrids compared with dsDNA permits accommodation of an additional base pair in the region between the primer 3'-end and the Fe2+-dependent cleavage position at the RNase H active site.
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PMID:Localization of the active site of HIV-1 reverse transcriptase-associated RNase H domain on a DNA template using site-specific generated hydroxyl radicals. 955 61


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