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

---

Query: EC:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A synthetic RNA oligonucleotide (15-mer) corresponding to the 3' end of the lysine tRNA primer was hybridized to single-stranded DNA containing the human immunodeficiency virus type 1 (HIV-1) primer-binding site and extended with a DNA polymerase. The resulting structures were used to study primer removal by the RNase H activity of HIV-1 reverse transcriptase. The initial cleavage event removes the RNA primer as a 14-mer and leaves a single ribonucleotide A residue bound to the 5' end of the DNA strand. This result explains the observation by several groups that HIV-1 circle junctions contain 4 bp that are not present in the integrated provirus instead of the predicted 3 bp. Subsequent cleavage events occur at other sites internal to the RNA molecule, and the ribonucleotide A residue on the end of the DNA strand is ultimately removed. Therefore, the biologically relevant cleavage that produces the 14-mer reflects the kinetics of the reaction as well as a specificity for nucleic acid sequence. When the RNA oligonucleotide alone was hybridized to the primer-binding site and tested as a substrate for HIV-1 RNase H, the cleavage pattern near the 3' end of the RNA was altered.
...
PMID:Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase. 137 87

Chemical modification of HIV-1 and HIV-2 (human immunodeficiency virus, types 1 and 2) reverse transcriptases (RT) with three thiol reactive compounds selectively inhibits the RNase H function of the enzyme. HIV-1 RT has 2 cysteines (at positions 38 and 280); HIV-2 RT has 3 (38, 280, 445). Both of the cysteines in HIV-1 RT are in the polymerase domain. To investigate the role of the cysteines in the structure and function of the HIV RTs, we have converted each cysteine to serine and made combinations of the mutations. Since HIV-1 RT has alanine at position 445, we have also substituted alanine for serine at this position in HIV-2 RT. Neither of the single mutations in HIV-1 RT nor the double mutation mimics the effects of the chemical modification. The serine 280 mutation has little effect on either polymerase or RNase H; the serine 38 mutation affects both activities, as does the 38/280 double mutant. The 38 and 280 serine mutations in HIV-2 RT resemble the equivalent mutations in HIV-1 RT. Substitution of serine or alanine at position 445 (which lies in the RNase H domain) diminishes, but does not abolish, the RNase H activity of HIV-2 without affecting polymerase activity. The RNase H activity of a mutant HIV-1 RT with serine at position 280 is completely resistant to inactivation by the three thiol reactive compounds we tested, which demonstrates that cysteine 280 is the critical residue. We suggest that the reason the mutation (cysteine 280 to serine) does not mimic the chemical modification is because the chemical modification produces a greater change in the structure of the protein. We also suggest that position 280 lies at or near the important points of contact between the RNase H and polymerase domains, so that chemical modification of this position, which lies within the polymerase domain, distorts the RNase H domain.
...
PMID:The effects of cysteine mutations on the reverse transcriptases of human immunodeficiency virus types 1 and 2. 137 Apr 63

We constructed a series of BspMI cassettes that simplify the introduction of specific point mutations in the polymerase domain of human immunodeficiency virus type 1 reverse transcriptase. A series of point mutants were constructed by using these cassette vectors. The RNA-dependent DNA polymerase and RNase H activities of 20 point mutations in the conserved portion of the polymerase domain were assayed. All the mutations analyzed are conservative substitutions of evolutionarily conserved amino acids. The mutations were divided into four classes. The first class has little effect on either polymerase or RNase H activity. The second class affects RNase H but not polymerase activity, while the third class has a normal RNase H activity with diminished polymerase activity. The fourth class affects both activities.
...
PMID:Cassette mutagenesis of the reverse transcriptase of human immunodeficiency virus type 1. 137 May 46

Reverse transcription of retroviral genomes requires the action of an RNase H for template switching and primer generation. In this report, we compare enzymatic properties of the RNase H associated with the reverse transcriptase (RT) from feline immunodeficiency virus (FIV) and that from human immunodeficiency virus (HIV). Both enzymes displayed substrate preference for poly[3H](rG) . poly(dC) hybird over poly[3H](rA) . poly(dT) and cation preference for Mg2+ over Mn2+. Activity of the FIV RNase H upon poly(rG) . poly(dC) produced hydrolysis products from 1 to 6 nucleotides in length, similar to that reported for HIV. Dextran sulfates were effective inhibitors of both the FIV and HIV RNase H and RT activities. Nearly identical inhibition constants (0.12 nM) were obtained for all enzyme activities with dextran sulfate 500,000, while different inhibition constants were observed with dextran sulfate 8,000. Our results suggest that FIV and HIV RTs contain a conserved region that is sensitive to the larger dextran sulfate and that dextran sulfate 8,000 may interact at a different site or by a different mechanism.
...
PMID:RNase H activity associated with reverse transcriptase from feline immunodeficiency virus. 137 May 49

We have studied a mutant Moloney murine leukemia virus with a deletion in reverse transcriptase (RT) which is predicted to make its RNase H domain resemble structurally that of human immunodeficiency virus RT. This deletion was based on improved RNase H homology alignments made possible by the recently solved three-dimensional structure for Escherichia coli RNase H. This mutant Moloney murine leukemia virus RT was fully active in the oligo(dT)-poly(rA) DNA polymerase assay and retained nearly all of wild-type RT's RNase H activity in an in situ RNase H gel assay. However, proviruses reconstructed to include this deletion were noninfectious. Minus-strand strong-stop DNA was made by the deletion mutant, but the amount of minus-strand translocation was intermediate to the very low level measured with RNase H-null virions and the high level seen with wild-type RT. The average length of translocated minus-strand DNA was shorter for the deletion mutant than for wild type, suggesting that mutations in the RNase H domain of RT also affect DNA polymerase activity.
...
PMID:Defects in Moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H. 137 May 51

We have demonstrated that the synthesis of cDNA by avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases can be prevented by oligonucleotides bound to the RNA template approximately 100 nucleotides remote from the 3' end of the primer. The RNA was truncated at the level of the antisense oligonucleotide-RNA duplex during the reverse transcription. The key role played by the reverse transcriptase-associated RNase H activity in the inhibition process was shown by the use of (i) inhibitors of RNase H (NaF or dAMP), (ii) Moloney murine leukemia virus reverse transcriptase devoid of RNase H activity, or (iii) alpha-analogues of oligomers that do not elicit RNase H-catalyzed RNA degradation. In all three cases the inhibitory effect was either reduced (NaF, dAMP) or totally abolished. However, an alpha-oligomer bound to the sequence immediately adjacent to the primer-binding site prevented reverse transcription. Therefore, initiation of polymerization can be blocked by means of an RNase H-independent mechanism, whereas arrest of a growing cDNA strand can be achieved only by an oligonucleotide mediating cleavage of the template RNA.
...
PMID:Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides. 137 May 86

In situ transcription (IST) was shown to be useful for the detection of human enteroviral RNA in cultured cells. A primer to detect a wide variety of enteroviral genomes and a coxsackievirus type B3 genome-specific primer were demonstrated to be efficient in IST assays. Transcription times greater than 10 to 30 min did not significantly improve the acquisition of a specific signal, whereas the signal-to-noise ratio decreased with time. Inclusion of actinomycin D to suppress DNA-dependent DNA polymerase activity in reverse transcriptase decreased the signal that was obtained without improving the signal-to-noise ratio. Use of RNase H-free murine leukemia virus reverse transcriptase in the IST reaction increased the signal versus that obtained by use of the avian myeloblastosis virus enzyme, which contains endogenous RNase H activity. Exogenous RNase H added to the transcription reaction ablated the signal. Background transcription because of poorly hybridized (mismatched) primers was reduced after primer hybridization and prior to the transcription reaction by rinsing fixed cells with 3 M tetramethylammonium chloride at temperatures which dissociate mismatched primer-template duplexes. The rapid detection time and the simplicity of application suggest that IST can be performed with a high specificity for the detection of enteroviral genomic sequences in cultured cells and may be more useful than in situ hybridization for the detection of enteroviral genomes.
...
PMID:Detection of enteroviruses in cell cultures by using in situ transcription. 137 Aug 49

Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA. This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities. In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme. The purified RT catalyzes a specific cleavage in HIV-1 RNA hybridized to tRNALys, the primer for HIV-1 reverse transcription. The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex. This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different. Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the HIV RNA-tRNALys complex. We refer to this unusual activity as RNase D. Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant HIV RT. The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column. Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity. Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNA-tRNALys at two distinct sites within the PBS sequence 5'-UGGCGCCCGA decreases ACAG decreases GGAC-3'. The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALys sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.
...
PMID:Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase. 137 Oct 14

The reverse transcriptases (RTs) from human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively) are relatively highly related yet there are several significant differences in their catalytic activities. In an attempt to relate these functional dissimilarities to the differences in amino acid sequences, we have employed a novel approach of constructing chimeric molecules composed of complementary amino acid sequences derived from the two HIV RTs. These recombinant proteins were analyzed for their enzymatic activities and for their sensitivity to tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2[1H]-one and thione (TIBO), which selectively inhibits only HIV-1 RT. The active chimeric RTs were used to map the TIBO binding site on the HIV-1 RT molecules and to localize the putative sequences responsible for the high RNase H activity of HIV-1 RT relative to that of HIV-2 RT. The results suggest that TIBO interacts with amino acid residues located around residue 200 within the DNA polymerase domain of HIV-1 RT which shows a relatively low similarity to HIV-2 RT. The difference in the RNase H activity maps to a position in the DNA polymerase domain rather than to the RNase H domain. Out of the 12 chimeric RTs generated, four were either fully active or hyperactive, three others lost most of their catalytic activities, and the rest were totally inactive. The pattern of catalytic activities of these hybrid proteins can be explained by a model for the initial folding of HIV RTs, which entails the formation of three distinct and independently folded regions. Each region can be formed by amino acid sequences derived exclusively from either HIV-1 RT or HIV-2 RT.
...
PMID:The catalytic functions of chimeric reverse transcriptases of human immunodeficiency viruses type 1 and type 2. 137 Dec 74

Phosphorothioate oligodeoxycytidine (S-dCn) was used as a model compound to examine the impact of the number of phosphorothioate linkages and their position on the inhibition of human DNA polymerases and RNase H in vitro. S-dCn with a chain length longer than 15 could inhibit human DNA polymerases and RNase H activities, in a linkage number-dependent manner. Longer oligomers were more potent inhibitors than shorter ones. Kinetic studies indicated that S-dC28 was a competitive inhibitor of DNA polymerase alpha and beta with respect to the DNA template, whereas it was a noncompetitive inhibitor of polymerases gamma and delta. S-dC28 was also a competitive inhibitor of RNase H1 and H2 with respect to RNA-DNA duplex. Susceptibility of these enzymes to inhibition by S-dC28 was in the order of delta approximately gamma greater than alpha greater than beta and RNase H1 greater than RNase H2. Structural-activity relationships were explored with a group of S-dC28 analogs that have phosphorothioate internucleotide linkages at various positions. The inhibitory effect depended on the total number of thioate linkages, rather than the position of the linkages within the oligomer or the chain length itself. No sequence specificity was found. In the presence of the complementary RNA, antisense phosphorothioates (S-oligos) exerted a biphasic effect on RNase H activity. At low concentrations S-oligos could enhance the cleavage of the RNA portion of S-oligo-RNA duplex, whereas at high concentrations (in excess of the complementary RNA) S-oligos could inhibit RNase H and protect the complementary RNA from degradation. Together, these results suggest that the non-sequence-specific inhibitory effect of S-oligos should be taken into consideration in designing antisense inhibitors. This inhibitory activity could be avoided by decreasing the number of phosphorothioate linkages at the backbone, and S-oligos of 15-20 residues are preferable in antisense molecule design.
...
PMID:Phosphorothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: implications for antisense technology. 137 82


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---