Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We demonstrated that unmodified and modified (phosphorothioate) oligonucleotides prevent cDNA synthesis by AMV or HIV reverse transcriptases. Antisense oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA fragment bound to the antisense oligonucleotide by the reverse transcriptase-associated RNase H activity. However, the phosphorothioate oligomer inhibited polymerization by binding to the AMV RT rather than to the template RNA, whereas there was no competitive binding of the phosphorothioate oligomer on the HIV RT during reverse transcription.
FEBS Lett 1993 Sep 13
PMID:Mechanisms of the inhibition of reverse transcription by unmodified and modified antisense oligonucleotides. 768 80

We examined the ribonuclease H (RNase H) specificity of human immunodeficiency virus reverse transcriptase (HIV-RT) using heteropolymeric RNAs hybridized to complementary DNAs. Experiments were performed in the presence of excess challenger polymer (poly(rA)-oligo(dT)) to reveal cleavages resulting from single enzyme binding events. Previous results suggested that initial RNase H directed cleavages were a fixed distance from a DNA primer terminus recessed on an RNA template, i.e. determined by the binding position of the polymerase active site. The influences of recessed RNA termini were not evaluated. In current experiments, RNAs that were 30, 42, or 50 nucleotides long were hybridized to the same 88 nucleotide long complementary DNA, such that the 5' terminal nucleotide of each RNA was hybridized to the 29th nucleotide from the 3' end of the DNA. In all three cases the RNA was initially cleaved between the 19th and 21st nucleotides from its 5' end. Thus, cleavage was not coordinated by the recessed 3' terminus of the RNA. Subsequent cleavages in either direction on the RNA were also observed. An insertion within the RNA that moved the preferred initial cut sequence 10 nucleotides further from the 5' end of the RNA decreased but did not abolish cleavage at the sequence. However, changing the nucleotide sequence in the region of the preferred cleavage either by the insertion experiment or mutagenesis did not significantly alter its capacity for cleavage. These results demonstrated a dominant position preference, plus a sequence priority. In another experiment, a 25 nucleotide long DNA was hybridized such that its 3' terminal nucleotide was 9 nucleotides from the 5' end of a 60 nucleotide complementary RNA. The preferred RNA cleavage sequence discussed above, was 10-14 nucleotides upstream of the 3' end of the DNA. However, initial cleavages occurred 17-20 nucleotides from the DNA 3' end, consistent with cleavage being coordinated by the recessed 3' terminus of the DNA primer.
Nucleic Acids Res 1993 Sep 11
PMID:Determinants of the RNase H cleavage specificity of human immunodeficiency virus reverse transcriptase. 769 1

The replicative cycle of the human immunodeficiency virus (HIV) is reviewed, and currently used and investigational agents directed against the virus are discussed. The first step in the replication of HIV is selective binding of the envelope glycoprotein to CD4 receptors located on T lymphocytes. The virion is then uncoated within the cytoplasm, yielding viral genomic RNA. Reverse transcriptase uses the viral RNA as a template to form single-stranded DNA, which is duplicated to form proviral DNA through the activity of ribonuclease H. Host RNA polymerases transcribe the integrated proviral DNA into messenger RNA, and there is subsequent translation to viral proteins. After translation, further modification of precursor polyproteins is necessary to produce functional peptides. The assembled virus then buds from the cell surface and invades other cells. Targets of drug intervention in the replicative cycle include (1) binding and entry, (2) reverse transcriptase, (3) transcription and translation, and (4) viral maturation and budding. Inhibitors of binding and entry include recombinant soluble CD4, immunoadhesins, peptide T, and hypericin. Nucleoside reverse-transcriptase inhibitors include zidovudine, didanosine, zalcitabine, and stavudine. Foscarnet, tetrahydroimidazobenzo-diazepinthione compounds, and nevirapine are some nonnucleoside reverse-transcriptase inhibitors. Inhibitors of transcription and translation include antagonists of the tat gene and GLQ223. Castanospermine, N-butyldeoxynojirimycin, and protease inhibitors interfere with viral maturation and budding. Drug combinations that have been or are being investigated include zidovudine plus interferon alfa, zidovudine plus zalcitabine, and zidovudine plus didanosine. Four agents currently have approved labeling for use against HIV infection: zidovudine, didanosine, zalcitabine, and stavudine. Monotherapy with zidovudine remains the treatment of first choice. Although progress has been made in developing drug therapies for HIV infection, more selective and more potent drugs are urgently needed. The best approach at present is to optimize the use of available agents, continue to investigate new therapies, and educate the public about prevention.
Am J Hosp Pharm 1994 Sep 15
PMID:Agents for treating human immunodeficiency virus infection. 775 75

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.
Protein Sci 1994 Sep
PMID:Equilibrium unfolding of Escherichia coli ribonuclease H: characterization of a partially folded state. 783 2

The 1235 residue herpes simplex virus DNA polymerase is a prototype alpha-like DNA polymerase and also an antiviral drug target. To investigate its organization, we mapped favored cleavage sites for seven proteases and identified three major classes of stable proteolytic fragments: 70-85-kDa N-terminal fragments, 50-70-kDa fragments that start near residues 600-700, and 12-kDa C-terminal fragments. In coimmunoprecipitation experiments, the first two classes of fragments remained associated; thus, cleavage in the center of the protein did not resolve structurally separate domains. In contrast, the 12-kDa C-terminal fragments did not remain associated with other fragments, suggesting a small separable C-terminal domain. The 70-85-kDa N-terminal fragments contained 3'-5' exonuclease and ribonuclease H activities; however, cleavage at the center of the molecule or near the C terminus appeared to destroy DNA polymerase activity. All three major classes of fragments bound DNA in DNA-cellulose chromatography and Southwestern blot analyses. The C-terminal fragments bound the viral polymerase processivity factor, UL42. The results map activities to regions of herpes simplex virus polymerase and suggest a model for its organization that may be pertinent to other DNA polymerases.
J Biol Chem 1994 Sep 09
PMID:Structural and functional organization of herpes simplex virus DNA polymerase investigated by limited proteolysis. 807 31

We have used protection against ribonuclease H to investigate the mechanisms by which U1 small nuclear ribonucleoprotein particles (snRNPs) determine the use of two alternative 5' splice sites. The initial binding of U1 snRNPs to alternative consensus splice sites was indiscriminate, and on a high proportion of pre-mRNA molecules both sites were occupied simultaneously. When the sites were close, this inhibited splicing. We propose that double occupancy leads to the use of the downstream site for splicing and that this is the cause of the proximity effect seen with strong alternative splice sites. This model predicts that splicing to an upstream site of any strength requires a low affinity of U1 snRNPs for the downstream site. This prediction was tested both by cleaving the 5' end of U1 snRNA and by altering the sequence of the downstream site of an adenovirus E1A gene. The enhancement of downstream 5' splice site use by splicing factor SF2/ASF appears to be mediated by an increase in the strength of U1 snRNP binding to all sites indiscriminately.
EMBO J 1993 Sep
PMID:Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF. 825 84

In MALME-3M human melanoma cells the polyamine analog N1,N12-bis(ethyl)spermine (BESPM) suppresses the key polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase, and increases the polyamine catabolizing enzyme, spermidine/spermine N1-acetyl-transferase (SSAT) by more than 200-fold. In the present study increases in SSAT activity in MALME-3M cells treated with 10 microM BESPM were found to be accompanied by a substantial (up to 45-fold) accumulation of SSAT mRNA. By Northern blot analysis three RNA transcripts were found to hybridize with the coding region of human SSAT cDNA: a minor high molecular weight (approximately 3.5 kilobases) species designated form A and two lower molecular weight species designated forms B and C (approximately 1.5 and approximately 1.3 kilobases, respectively). Form A increased uniformly during BESPM treatment and was most obvious in nuclear RNA preparations. On the basis of size similarity to the transcribing region of the gene and hybridization with the coding region of SSAT cDNA and its prevalence in nuclear mRNA preparations, form A is thought to represent precursor SSAT RNA. Form C is present in control cells and increases steadily during treatment, whereas form B increases transiently during early treatment (1-3 h). By RNase H digestion assay, form B was found to have a 200-base pair longer poly(A) tract and as such may represent a precursor to form C. Accumulation of SSAT mRNA was found to be a result of increased gene transcription and stabilization of SSAT mRNA. Nuclear run-on studies indicated a 2-4-fold increase in the transcription rate of the SSAT gene. As indicated by actinomycin D studies, the SSAT mRNA half-life increased with BESPM treatment from 17 to 64 h. The natural polyamine, spermine, also increased SSAT mRNA (5.5-fold at 24 h) and behaved similarly to BESPM in inducing the appearance of the same three transcript forms. The polyamine was much less effective than the analog at increasing enzyme activity. Lowering intracellular polyamine pools with inhibitors of biosynthesis decreased basal SSAT mRNA levels by at least 70% indicating, that the gene can be down-regulated as well as up-regulated by polyamines. These findings indicate that SSAT represents a unique example of gene expression being positively influenced at the RNA level by polyamines and their analogs.
J Biol Chem 1993 Sep 05
PMID:Polyamine and polyamine analog regulation of spermidine/spermine N1-acetyltransferase in MALME-3M human melanoma cells. 836 Jan 94

Two families of retrotransposons, Tf1 and Tf2, have been isolated from the fission yeast, Schizosaccharomyces pombe. We report here the nucleotide (nt) sequence of a Tf2 element, the only retrotransposon family known from the commonly used laboratory strains, 972 and 975, and their derivatives. The total nt sequence of Tf2 was derived from the complete sequence of the coding region and 3' long terminal repeat (LTR) of randomly cloned element Tf2-1, and from a full 5' LTR and approximately one-third of the open reading frame (ORF) of Tf2-43, a Tf2 element found in the head-to-head orientation adjacent to the Sz. pombe rpb6 gene. The two Tf2 sequences are nearly identical and both of them contain a single ORF encoding a protein with regions of sequence similar to protease, reverse transcriptase, RNase H (RH) and integrase from other retrotransposons and retroviruses. Sequence comparisons between Tf1 and Tf2 indicate an extreme divergence of the putative capsid protein-encoding regions of these two elements, as well as divergence of a segment of the LTR, but otherwise virtually identical sequence.
Gene 1993 Sep 06
PMID:Sequence analysis of closely related retrotransposon families from fission yeast. 839 47

To clone new replication origin(s) activated under RNase H-defective (rnh-) conditions in Escherichia coli cells, whole chromosomal DNA digested with EcoRI was to with a Kmr DNA fragment and transformed into an rnh- derivative host. From the Kmr transformants, we obtained eight kinds of plasmid-like DNA, each of which contained a specific DNA fragment, termed "Hot", derived from the E. coli genome. Seven of the Hot DNAs (HotA-G) mapped to various sites within a narrow DNA replication termination region (about 280 kb), without any particular selection. Because Hot DNA could not be transformed into a mutant strain in which the corresponding Hot region had been deleted from the chromosome, the Hot DNA, though obtained as covalently closed circular (ccc) DNA, must have arisen by excision from the host chromosome into which it had initially integrated, rather than by autonomous replication of the transformed species. While Hot DNA does not have a weak replication origin it does have a strong recombinational hotspot active in the absence of RNase H. This notion is supported by the finding that Chi activity was present on all Hot DNAs tested and no Hot-positive clone without Chi activity was obtained, with the exception of a DNA clone carrying the dif site.
Mol Gen Genet 1993 Sep
PMID:Specific chromosomal sites enhancing homologous recombination in Escherichia coli mutants defective in RNase H. 841 78

Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.
J Cell Biochem 1995 Sep
PMID:Further characterization of the human cell multiprotein DNA replication complex. 853 May 40


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