Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Phosphorothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: implications for antisense technology. 137 82

A DNA fragment encoding Ribonuclease H (EC 3. 1.26.4) was isolated from an extreme thermophilic bacterium, Thermus thermophilus HB8, by its ability to complement the temperature-sensitive growth of an Escherichia coli rnhA deficient mutant. The primary amino acid sequence showed 56% similarity to that of E. coli RNase HI but little or no homology to E. coli RNase HII. Enzymes derived from thermophilic organisms tend to have fewer cysteines than their bacterial counterparts. However, T. thermophilus RNase H has one more cysteine than its E. coli homologue. Stability of the RNase H in extracts of T. thermophilus to elevated temperatures was the same for the protein expressed in E. coli. T. thermophilus RNase H should, therefore, be a useful tool for editing RNA-DNA hybrid molecules at higher temperatures and may also be stable enough to be used in a cyclical process. It was suggested that regulation of expression of the RNase H may be different from that of E. coli. RNase HI.
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PMID:Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme thermophile Thermus thermophilus HB8: a thermostable RNase H can functionally replace the Escherichia coli enzyme in vivo. 165 14

The major ribonuclease H from K562 human erythroleukemia cells has been purified more than 4,000-fold. This RNase H, now termed RNase H1, is an endoribonuclease whose products contain 5'-phosphoryl and 3'-hydroxyl termini. The enzyme has a native molecular weight of 89,000 based on its sedimentation and diffusion coefficients. Human RNase H1 has an absolute requirement for a divalent cation. Maximal activity is obtained with either 10 mM Mg2+, 5 mM Co2+, or 0.5 mM Mn2+. The pH optimum is between 8.0 and 8.5 in the presence of 10 mM Mg2+. The isoelectric point is 6.4. RNase H1 lacks double-stranded and single-stranded RNase and DNase activities, and it will not hydrolyze the DNA moiety of an RNA.DNA heteroduplex. Unlike the Escherichia coli enzyme, which requires a heteroduplex that contains at least four consecutive ribonucleotides for activity, human RNase H1 can hydrolyze a DNA.RNA.DNA/DNA heteroduplex that contains a single ribonucleotide. Cleavage occurs at the 5' phosphodiester of this residue. This substrate specificity suggests that human RNase H1 could play a role in ribonucleotide excision from genomic DNA during replication.
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PMID:Ribonuclease H from K562 human erythroleukemia cells. Purification, characterization, and substrate specificity. 170 18

An additional RNase H (EC 3.1.26.4), RNase HII, has been isolated from Escherichia coli K-12. By screening a library of E. coli DNA for clones that suppressed RNase H deficiency of an E. coli rnh mutant, a clone was obtained that produced a protein with RNase H activity. The overexpressed RNase HIII protein in E. coli was purified to near homogeneity and exhibited a strong preference for the ribonucleotide moiety of RNA-DNA hybrid as substrate. The terminal 11 amino acids were determined and were identical to those predicted from the nucleotide sequence. The rnhB gene, which encodes RNase HII, was distinct from rnhA by its map position (4.5 min on E. coli genetic map, between lpxB and dnaE) and by the lack of significant amino acid sequence similarity. The presence of a second RNase H in E. coli indicates that multiple RNase H genes per genome is a general feature of a general feature of a wide variety of organisms.
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PMID:Isolation and characterization of a second RNase H (RNase HII) of Escherichia coli K-12 encoded by the rnhB gene. 217 91

Eukaryotic ribonucleases H of known sequence are composed of an RNase H domain similar in size and sequence to that of Escherichia coli RNase HI and additional domains of unknown function. The RNase H1 of Saccharomyces cerevisiae has such an RNase H domain at its C-terminus. Here we show that the N-terminal non-RNase H portion of the yeast RNase H1 binds tightly to double-stranded RNA (dsRNA) and RNA-DNA hybrids even in the absence of the RNase H domain. Two copies of a sequence with limited similarity to the dsRNA-binding motif are present in this N-terminus. When the first of these sequences is altered, the protein no longer binds tightly to dsRNA and exhibits an increase in RNase H activity. Unlike other dsRNA-binding proteins, increasing the Mg2+ concentration from 0.5 mM to 5 mM inhibits binding of RNase H1 to dsRNA; yet a protein missing the RNase H domain binds strongly to dsRNA even at the higher Mg2+ concentration. These results suggest that binding to dsRNA and RNase H activity are mutually exclusive, and the Mg2+ concentration is critical for switching between the activities. Changes in the Mg2+ concentration or proteolytic severing of the dsRNA-binding domain could alter the activity or location of the RNase H and may govern access of the enzyme to the substrate. Sequences similar to the dsRNA-binding motif are present in other eukaryotic RNases H and the transactivating protein of cauliflower mosaic virus, suggesting that these proteins may also bind to dsRNA.
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PMID:The non-RNase H domain of Saccharomyces cerevisiae RNase H1 binds double-stranded RNA: magnesium modulates the switch between double-stranded RNA binding and RNase H activity. 748 97

Bacterial reverse transcriptase is responsible for the synthesis of multicopy single-stranded DNA (msDNA). Reverse transcriptases from retron-Ec73 and retron-Ec107 do not contain an RNase H domain. Cellular RNase H is therefore considered to be required to make the mature form of msDNA. We found that RNase HI, but not RNase HII, is required for the production of the mature form of both msDNAs.
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PMID:The role of ribonuclease H in multicopy single-stranded DNA synthesis in retron-Ec73 and retron-Ec107 of Escherichia coli. 752 2

A ribonuclease H activity from human placenta has been separated by ion exchange chromatography from the major RNase HI enzyme. Additional chromatographic steps allowed further purification, more than 3,000 fold compared to the crude extract in which it represents about 15% of the total RNase H activity. The enzyme requires Mg2+ ions for its activity, is strongly inhibited by the addition of Mn2+ ions or other divalent transition metal ions, and exhibits a pH optimum between 8.5 and 9. It shows a strong sensitivity to the SH-blocking agent N-ethylmaleimide. It has a strict specificity for double-stranded RNA-DNA duplexes and exhibits neither single-stranded nor double-stranded RNase (or DNase) activities. Therefore, this enzyme displays the characteristics of class II RNase H and is now termed RNase HII. Renaturation gel assays and gel filtration experiments proved a monomeric structure for the active enzyme with a native molecular weight of about 33 kDa. The human RNase HII acts as an endonuclease and releases oligoribonucleotides with 3'-OH and 5'-phosphate ends. It is therefore a candidate for the RNase H-mediated effect of antisense oligodeoxynucleotides.
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PMID:Purification and characterization of human ribonuclease HII. 781 13

We have cloned and functionally characterized the RNase H1 gene from D. melanogaster. The longest open reading frame consists of 5 exons that encode a 333 amino acid protein with a molecular mass of 37.1 kDa. This is the first demonstration of specific nuclease activity of a cloned RNase gene from a multicellular higher eukaryote. No additional proteins or cofactors are required for this nuclease activity. Comparison of Drosophila RNase H1 amino acid sequence to that of other cellular eukaryotic homologs reveals the presence of three evolutionarily distinct domains. The N- and C-terminal conserved domains are connected by a highly variable domain. The C-terminal domain has high amino acid similarity to bacterial RNase HI and the RNase H domain of retroviral reverse transcriptase, while the N-terminus, of unknown function, is similar to the P6 translational activator of caulimoviruses.
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PMID:Functional characterization of RNase H1 from Drosophila melanogaster. 939 56

We cloned the Saccharomyces cerevisiae homologue of mammalian RNase HI, which itself is related to the prokaryotic RNase HII, an enzyme of unknown function and previously described as having minor activity in Escherichia coli. Expression of the corresponding yeast 35 kDa protein (named by us RNase H(35)) in E. coli and immunological analysis proves a close evolutionary relationship to mammalian RNase HI. Deletion of the gene (called RNH35) from the yeast genome leads to an about 75% decrease of RNase H activity in preparations from the mutated, still viable cells. Sequence comparison discriminates this new yeast RNase H from earlier described yeast enzymes, RNase H(70) and RNase HI.
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PMID:Yeast RNase H(35) is the counterpart of the mammalian RNase HI, and is evolutionarily related to prokaryotic RNase HII. 946 32

Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif. Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif. A very similar amino acid sequence is present in caulimovirus ORF VI proteins. The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic. Point mutations, insertions and deletions indicated that integrity of the motif is important for binding. However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well. Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence. The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.
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PMID:A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs. 951 60


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