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)

Two constituent protein domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase were expressed separately and purified to homogeneity. The N-terminal domain (p51) behaves as a monomeric protein exhibiting salt-sensitive DNA polymerase activity. The C-terminal domain (p15) on its own has no detectable RNase H activity. However, the combination of both isolated p51 and p15 in vitro leads to reconstitution of RNase H activity on a defined substrate. These results demonstrate that domains of HIV-1 reverse transcriptase are functionally interdependent to a much higher degree than in the case of reverse transcriptase from Moloney murine leukemia virus.
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PMID:Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase. 170 27

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
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PMID:Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. 171 5

A sedimentable complex of enzymes for DNA synthesis was partially purified from the combined low-salt nuclear extract-postmicrosomal supernatant solution of HeLa cell homogenates by poly(ethylene glycol) precipitation in the presence of 2 M KCl, discontinuous gradient centrifugation, Q-Sepharose chromatography, and velocity gradient centrifugation. In addition to the previously described 640-kDa multiprotein DNA polymerase alpha-primase complex [Vishwanatha et al. (1986) J. Biol. Chem. 261, 6619-6628], the enzyme complex also has associated topoisomerase I, DNA-dependent ATPase, RNase H, DNA ligase, a simian virus 40 origin recognition, dA/dT sequence binding protein [Malkas & Baril (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 70-74], and proliferating cell nuclear antigen. Essentially all of the T antigen dependent simian virus 40 in vitro replication activity in the combined nuclear extract-postmicrosomal supernatant solution resides with the sedimentable complex of enzymes for DNA synthesis. Sedimentation analysis on a 10-35% glycerol gradient in the presence of 0.5 M KCl indicates that the enzyme complex is 21S. The associated enzymes for DNA synthesis and in vitro simian virus 40 replication activity cofractionate throughout the purification of the 21S complex. The DNA polymerase and in vitro simian virus 40 replication activities are both inhibited by monoclonal antibody (SJK 132-20) to human DNA polymerase alpha and by 5-10 microM butylphenyl-dGTP, indicating that the association of DNA polymerase alpha with the 21S enzyme complex is essential for the initiation of SV40 DNA replication in vitro.
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PMID:A 21S enzyme complex from HeLa cells that functions in simian virus 40 DNA replication in vitro. 216 68

The avian RNA tumour virus structural protein p12 was purified from avian myeloblastosis virus (AMV) by nucleic acid affinity chromatography to apparent homogeneity as judged from SDS--polyacrylamide gel electrophoresis. A filter binding assay was used for the identification of p12. High concentrations of p12 precipitated nucleic acids out of solution in the absence of MgCl2. Binding of p12 to single-stranded nucleic acids protected them from digestion with nucleases and resulted in a hyperchromic effect. These phenomena were reversible in the presence of salt. The affinity of p12 to nucleic acids was determined by competing for the binding of p12 to denatured radioactive DNA by various other nuclei acids. It was found that p12 bound preferentially to single-stranded nucleic acids and showed a higher affinity to poly(rI) than to poly(rC) and poly(rA). Purified RNA-dependent DNA polymerase activity from AMV was stimulated up to sixfold by p12, depending on the template. Solubilization of RNA in RNA--DNA hybrids by RNase H was inhibited in the presence of p12.
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PMID:Properties of the avian viral protein p12. 616 96

Rat liver nuclei were isolated in aqueous solutions of low ionic strength or anhydrous glycerol. The presence of ribonuclease H (RNase H) [EC 3.1.4.34] activity in the cytoplasm is due to extraction of the nuclear enzyme by buffer and inorganic salts. Two forms of RNase H were separated from rat liver nuclei by affinity chromatography using a DNA-cellulose column. When the RNase H in the wash solution of nuclei with 0.3 M sucrose and in nuclear solution extracted with 0.15 M NaCl were fractionated on a single-stranded DNA-cellulose column, two peaks corresponding to Mn2+- and Mg2+-dependent RNases H were eluted at 0.1 M and 0.2 M NaCl, respectively, and a peak having both RNase H activities was recovered in the wash-through fraction from the column. Among the enzymes from these two fractions in the nuclei, the activity of the Mg2+-dependent RNase H which binds to DNA-cellulose increased several-fold within 24 h of a single injection of thioacetamide. The activities of Mg2+-dependent RNase H extracted with higher-salt solution from the nuclei and recovered in the flow-through fraction from the DNA-cellulose column and the Mn2+-dependent RNase H activities were relatively unaffected by an injection of thioacetamide.
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PMID:Nuclear location of ribonuclease H and increased level of magnesium-dependent ribonuclease H in rat liver on thioacetamide treatment. 627 76

At an early purification stage, DNA polymerase alpha holoenzyme from calf thymus can be separated into four different forms by chromatography on DEAE-cellulose. All four enzyme forms (termed A, B, C, and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase alpha, a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3'-5' exonuclease, and RNase H activity. Peaks B, C, and D all contain, together with DNA polymerase alpha, activities of primase and DNA topoisomerase type II. Furthermore, peak B is enriched in an RNase H, and peaks C and D are enriched in a 3'-5' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the four peaks gave evidence of unexpectedly large forms of DNA polymerase alpha (greater than 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase alpha. Peak C is more resistant to inhibition by salt and spermidine than the other three enzyme forms. These results suggest the existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C, and D). Finally, the salt-resistant C form might represent a functional DNA polymerase alpha holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.
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PMID:Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork. 658 75

Replicating DNA of human adenovirus type 2, identified as partly single-stranded viral DNA in which [3H]thymidine is readily incorporated, was found to be separated into two fractions by chromatography on hydroxyapatite. Whereas one of the these fractions was eluted with 180 mM phosphate, the other one was eluted at the same concentration, 240 mM, as fully double-stranded DNA. The physical properties of the 180 and 240 mM fractions, in particular their buoyant densities in solutions of CsCl and Cs2SO4, were compared both before and after treatment by various enzymes such as Neurospora crassa nuclease, pancreatic ribonuclease, ribonuclease H and the Klenow fragment of DNA polymerase I of Escherichia coli, used alone or in various combinations. Unlike the 240 mM fraction, the 180 mM fraction was found to include a substantial amount of single-stranded DNA, some of it being hydrogen-bonded to RNA. Both of these features confer to the 180 mM fraction the high buoyant density in cesium salt solution which was described, for several adenoviruses, as one of the characteristic properties of replicating DNA.
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PMID:Two classes of replicating molecules of adenovirus type 2 DNA. 724 95

The mechanism of human immunodeficiency virus reverse transcriptase-catalyzed strand transfer synthesis (i.e. switching of the primer to a new template) from internal regions of natural sequence RNA was investigated. The system consisted of a 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide used to initiate synthesis. DNA oligonucleotides with homology to internal regions of the donor were used as acceptor templates. In reactions performed in the absence of acceptor template, a prominent DNA synthesis product 75 nucleotides in length resulting from pausing DNA synthesis within the homology zone was observed. Prominent donor RNA degradation products of 47 or 54 nucleotides were also observed, in reactions with 80 or 150 mM KCl, respectively. The lengths indicated a potential 13- or 20-nucleotide long, respectively, complementary region between the DNA and RNAs. The 54-, but not the 47-, nucleotide RNA was susceptible to Escherichia coli RNase H, indicating that the DNA was annealed only to the 54-mer. When acceptor was added, a portion of the 75-nucleotide DNA was chased into transfer product at both salt concentrations, and a portion of the 54-mer RNA became resistant to E. coli RNase H. Evidently, this donor RNA was annealed to the 75-nucleotide long DNA but could be actively displaced by the acceptor. Overall, these observations support two mechanisms for transfer. In one, the pause site-specific DNA dissociates from the donor template before transferring. In the other, the acceptor actively displaces the DNA from the donor.
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PMID:The mechanism of human immunodeficiency virus reverse transcriptase-catalyzed strand transfer from internal regions of heteropolymeric RNA templates. 750 52

The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine. RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell. Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66. The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA. Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge. Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase. Homology data suggest that such a bridge may be present in other lentiviruses. The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity.
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PMID:Disruption of a salt bridge between Asp 488 and Lys 465 in HIV-1 reverse transcriptase alters its proteolytic processing and polymerase activity. 769 May 4

The recombinant 94 kDa Thermus thermophilus DNA polymerase (rTth pol) was found to release [33P]UMP when incubated with a RNA.DNA hybrid containing a [33P]UMP-labeled RNA strand. The RNase H activity was optimally active in the presence of low monovalent salt concentrations and when Mn2+ was used as the divalent cation activator. RNase H activity also was observed when Mg2+ replaced the Mn2+, but to a much lesser extent. A 60 nucleotide long, 5'- or 3'-radiolabeled RNA or DNA oligomer hybridized to a complementary DNA oligomer was used to determine the mode of digestion. The radiolabeled RNA.DNA hybrid or DNA.DNA duplex was incubated with rTth pol using various metal ion conditions and different incubation times. The DNA.DNA duplex showed very little enzymatic cleavage by rTth pol regardless of the Mn2+ or Mg2+ concentration. However, nearly complete digestion of the RNA.DNA hybrid was observed over a wide Mn2+ concentration range, thus demonstrating a preferential degradation of the RNA.DNA hybrid vs the DNA.DNA duplex. Time course reactions of the enzymatic digestion of the 3'-labeled RNA.DNA hybrid or DNA.DNA duplex by rTth pol indicated that digestion of the substrates occurred exonucleolytically in the 5'-->3' direction.
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PMID:Properties of the 5'-->3' exonuclease/ribonuclease H activity of Thermus thermophilus DNA polymerase. 771 Oct 21


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