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)

Replication of retroviral RNA into double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract, PPT, by the reverse transcriptase (RT). The PPT is highly conserved among the known HIV-1 retroviral isolates. It occurs twice, once within the coding region of the integrase and the other one adjacent to the 3' LTR. The data presented show that two antisense oligonucleotides, a 20-mer and a 40-mer, complementary to the PPT induce complete blocks of DNA synthesis whereas an antisense oligonucleotide outside the PPT is only slightly inhibitory. Previously polypurine sequences have been used by several groups for triplex-formation. During replication the HIV-polypurine tract, PPT, is present in a RNA-DNA hybrid. Therefore triple-helix formation consisting of RNA-DNA and a third DNA strand covering the PPT region was tested here by protection against RNase H cleavage in vitro. Incubation with a pyrimidine oligonucleotide in parallel orientation to the PPT-RNA shows some protection. GT-pyrimidine-purine mixed oligonucleotides (25-mer) led to protection against RNase H up to 50% independent of their orientation. The data suggest that triple-helix formation may have taken place with the PPT in vitro. Therefore, this highly conserved structure may prove useful in nucleic acid based anti-viral therapy with antisense or triple-helix approaches. Furthermore, the influence of HIV-1 nucleocapsid (NC) protein, NCp15, on reverse transcription is reported. The data show a two- to three-fold stimulatory effect of the NCp15 on RNA directed DNA synthesis.
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PMID:The polypurine tract, PPT, of HIV as target for antisense and triple-helix-forming oligonucleotides. 768 36

We have exploited the sole tryptophan residue (Trp535) in the ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) to study features of the isolated polypeptide (p15 RNase H) by fluorescence spectroscopy. Incubation of purified p15 RNase H with a synthetic RNA/DNA hybrid was accompanied by an alteration in Trp535 fluorescence intensity. This property was used to determine an apparent binding constant (Kapp) of 3.5 x 10(6) M-1 for p15 RNase H complexed with poly(rA)/oligo(dT)12-18 and an occluded site size of 4 nucleotides. A cooperativity coefficient (omega) of 910 was also determined which indicated that nearly three logs of the Kapp were due to cooperativity effects. Recombinant p15 RNase H preparations containing mutations at position 478 (Glu478-->Gln478) or 539 (His539 -->Phe539), which are highly conserved between bacterial and retroviral RNases H, were also analyzed. Under the same conditions, these mutants failed to bind the RNA/DNA hybrid, although they were structurally similar to the wild type polypeptide. Fluorescence spectroscopy thus appears to be an alternative and sensitive means of analyzing functional properties of the purified RNase H domain of HIV-1 RT under a variety of conditions.
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PMID:Fluorimetric analysis of recombinant p15 HIV-1 ribonuclease H. 768 4

Mag is a retrotransposon found as an insert in the Sericin 2 gene. It is present in a few copies--4 to 15--dispersed in the genome of different strains of Bombyx mori as well as in Bombyx mandarina. Flanked by a 5 bp target sequence with no sequence specificity, it is bordered by direct repeats of 77 nucleotides. Despite their unusual short size, these terminal repeats and their immediately adjacent sequences present all the signals necessary for transcription into genomic RNA and for reverse transcription. Mag contains two overlapping open reading frames which are organized as the gag and pol genes of retroviruses and encode putative nucleic acid binding peptide, protease, reverse transcriptase, RNase H and endonuclease in this order. Sequence comparison of these proteins places mag within the gypsy group of LTR retrotransposons next to the echinoderm element SURL.
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PMID:Structural features of mag, a gypsy-like retrotransposon of Bombyx mori, with unusual short terminal repeats. 781 9

We have examined the equilibrium unfolding of Escherichia coli ribonuclease HI (RNase H), a member of a family of enzymes that cleaves RNA from RNA:DNA hybrids. A completely synthetic gene was constructed that expresses a variant of the wild-type sequence with all 3 cysteines replaced with alanine. The resulting recombinant protein is active and folds reversibly. Denaturation studies monitored by circular dichroism and tryptophan fluorescence yield coincident curves that suggest the equilibrium unfolding reaction is a 2-state process. Acid denaturation, however, reveals a cooperative transition at approximately pH 1.8 to a partially folded state. This acid state can be further denatured in a reversible manner by the addition of heat or urea as monitored by either CD or tryptophan fluorescence. Analytical ultracentrifugation studies indicate that the acid state of RNase H is both compact and monomeric. Although compact, the acid state does not resemble the native protein: the acid state displays a near-UV CD spectrum similar to the unfolded state and binds to and enhances the fluorescence of the dye 1-anilinonaphthalene, 8-sulfonate much more than either the native or unfolded states. Therefore, the acid state of E. coli RNase H has the characteristics of a molten globule: it retains a high degree of secondary structure, remains compact, yet does not appear to contain a tightly packed core.
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PMID:Equilibrium unfolding of Escherichia coli ribonuclease H: characterization of a partially folded state. 783 2

A repeated DNA sequence used for epidemiological studies of the human opportunistic pathogen Aspergillus fumigatus has been characterized. It is a retroelement of 6914 bp in length, bounded by long terminal repeats of 282 bp, with sequence and features characteristic of retroviruses and retrotransposons. A 5 bp duplication site was found at its borders. This element, designated Afut1, encodes amino acid sequences homologous to the reverse transcriptase, RNase H and endonuclease encoded by the pol genes of retroelements. Comparison of the peptidic sequences with other putative polypeptides of fungal LTR retrotransposons showed that Afut1 is a member of the gypsy group. This is the first report of a transposable element in A.fumigatus. Afut1 is a defective element: the putative coding domains contain multiple stop codons due exclusively to transitions from C:G to T:A.
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PMID:Afut1, a retrotransposon-like element from Aspergillus fumigatus. 862 74

A previously described self-complementary oligodeoxynucleotide termed triplex-forming oligodeoxynucleotide (TFO A), 54 bases in length, designed against the polypurine tract of HIV-1 RNA, inhibited viral replication at a 1 to 3 microM concentration in acutely infected cells, whereas antisense and scrambled sequence oligodeoxynucleotides were ineffective. Three HIV-1 viral isolates from patients of clinical categories A1, B, and C3 were transmitted to peripheral blood mononuclear cells and tested for production of p24 antigen and syncytium formation in the absence and in the presence of either TFO A or a control oligodeoxynucleotide of randomized sequence. No p24 antigen or syncytia were detected for up to 30 days when TFO A was added to the cells. Viability of the cells was found not to be affected by the drugs compared to controls within 2 weeks. Analysis of viral DNA synthesis by PCR for the LTR and gag gene indicated no DNA signal, suggesting that TFO A affects viral replication before formation of a DNA provirus. Measurements of the stability of TFO A indicate a half-life of about 2 hr. A two-dimensional computer fold analysis of TFO A suggested a self-complementary hairpin-loop configuration with GC-rich stems and single-stranded 5' and 3' ends. Since intracellular triplex formation may not be an efficient process, the observed inhibitory effect may be due to a direct inhibition of the RT and RNase H enzyme activities by the oligodeoxynucleotide. However, a triple-helix effect on the incoming RNA may play a role as well.
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PMID:Inhibition of replication of fresh HIV type 1 patient isolates by a polypurine tract-specific self-complementary oligodeoxynucleotide. 884 20

We have isolated and characterized a new human endogenous provirus, which is closely related to the human retrovirus S71, but unlike S71 has a full-length pol gene. Two degenerate oligonucleotide primers based on highly conserved motifs within the active sites of two retroviral proteins (the protease and reverse transcriptase) were designed and used for PCR. An amplified product of 847 bp in length, which showed significant homology to protease and reverse transcriptase of several retroviruses, was used for high stringency hybridization with a human genomic library. The MuLV-related endogenous retrovirus sequence, designated HC2, was isolated and completely sequenced. HC2 is a provirus with complete gag and pol genes and a 3' LTR; the 5' LTR and env gene are missing. The gag and pol genes appear complete, since they contain sequences homologous to the matrix protein, capsid protein, and nucleocapsid protein of gag and to the protease, reverse transcriptase, tether, RNase H, and integrase of pol. Phylogenetic analysis suggests that although HC2 and S71 are MuLV-related retroviruses, their characters are quite distinct, being placed outside of a clade containing most of the previously characterized MuLV-related retroviruses such as GaLV, FeLV, BaEV, and SSV/SSAV.
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PMID:Human endogenous retrovirus HC2 is a new member of the S71 retroviral subgroup with a full-length pol gene. 894 25

RNA-DNA hybrid model substrates which mimic an intermediate of Moloney murine leukemia virus (M-MuLV) reverse transcription at the stage where the tRNAPro is removed were constructed. This substrate was used to assay the ability of M-MuLV reverse transcriptase (RT) to cleave the RNA portion of the substrate. The cleavage specificities of the cognate M-MuLV RT and the heterologous enzyme from the human immunodeficiency virus type-1 (HIV-1) were compared. M-MuLV and HIV-1 RT recognize and cleave the RNA at distinct positions. The site of the initial RNase H cleavage in vitro was determined using 3' end nearest neighbor analysis of the initial cleavage product. M-MuLV RT/RNase H removed the model tRNAPro between the terminal ribo-A and ribo-C, resulting in a terminal ribo-A attached to the viral DNA, whereas HIV-1 RT/RNase H was shown to cleave at the RNA-DNA junction. Analysis of the DNA over time indicated that the ribo-A is subsequently removed by M-MuLV RT. In vivo analysis from double-LTR circle junctions illustrated that 16 of the 23 clones isolated possessed the predicted junction if complete removal of the tRNA primer were to occur. The predicted junction for complete removal of the tRNA primer was CATT-AATG. One aberrant circle junction was isolated which could result from the use of an alternative primer. In contrast with HIV, no M-MuLV circle junctions were isolated which indicated processing of a single-LTR terminus by integrase. Analysis from in vivo and in vitro studies indicate that the M-MuLV tRNAPro primer is completely removed after plus-strand strong-stop synthesis.
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PMID:RNase H cleavage of tRNAPro mediated by M-MuLV and HIV-1 reverse transcriptases. 912 56

The BARE-1 copia-like retrotransposon constitutes nearly 7% of the barley (Hordeum vulgare L.) genome as a family of more than 2 x 10(4) mostly full-length copies dispersed on all chromosomes. BARE-1 elements are transcribed in barley tissues from promoters within the LTR (long terminal repeat). The predicted, translated polyprotein contains conserved domains for GAG, aspartic proteinase, integrase, reverse-transcriptase, and RNase H. Here, we have used inverse PCR with LTR-based primers to establish the consensus sequences for the terminal region of the LTR, the external dinucleotides of the cDNA integration intermediate, and the minus- and plus-strand priming sites. These key functional entities are well-conserved in the BARE-1 family, including wheat Wis2, but differ from those of other plant retrotransposons. The target site duplication was established as 5 bp. Of the 13 integration sites identified here, 8 were other BARE-1 elements and 1 another retrotransposon; 59% of the total 17 identified BARE-1 insertion sites are retrotransposons. This nested insertion pattern may represent a basic feature of plant retrotransposons.
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PMID:BARE-1 insertion site preferences and evolutionary conservation of RNA and cDNA processing sites. 944 Feb 75

Retroviruses and their relatives, the LTR-retrotransposons, possess an integrase protein (IN) that is required for the insertion of reverse transcripts into the genome of host cells. Schizosaccharomyces pombe is the host of Tf1, an LTR-retrotransposon with integration activity that can be studied by using techniques of yeast genetics. In this study, we sought to identify amino acid substitutions in Tf1 that specifically affected the integration step of transposition. In addition to seeking amino acid substitutions in IN, we also explored the possibility that other Tf1 proteins contributed to integration. By comparing the results of genetic assays that monitored both transposition and reverse transcription, we were able to seek point mutations throughout Tf1 that blocked transposition but not the synthesis of reverse transcripts. These mutant versions of Tf1 were candidates of elements that possessed defects in the integration step of transposition. Five mutations in Tf1 that resulted in low levels of integration were found to be located in the IN protein: two substitutions in the N-terminal Zn domain, two in the catalytic core, and one in the C-terminal domain. These results suggested that each of the three IN domains was required for Tf1 transposition. The potential role of these five amino acid residues in the function of IN is discussed. Two of the mutations that reduced integration mapped to the RNase H (RH) domain of Tf1 reverse transcriptase. The Tf1 elements with the RH mutations produced high levels of reverse transcripts, as determined by recombination and DNA blot analysis. These results indicated that the RH of Tf1 possesses a function critical for transposition that is independent of the accumulation of reverse transcripts.
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PMID:The application of a homologous recombination assay revealed amino acid residues in an LTR-retrotransposon that were critical for integration. 944 33


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