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1.
EMBO J ; 37(15)2018 08 01.
Article in English | MEDLINE | ID: mdl-29959219

ABSTRACT

Long INterspersed Element class 1 (LINE-1) elements are a type of abundant retrotransposons active in mammalian genomes. An average human genome contains ~100 retrotransposition-competent LINE-1s, whose activity is influenced by the combined action of cellular repressors and activators. TREX1, SAMHD1 and ADAR1 are known LINE-1 repressors and when mutated cause the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). Mutations in RNase H2 are the most common cause of AGS, and its activity was proposed to similarly control LINE-1 retrotransposition. It has therefore been suggested that increased LINE-1 activity may be the cause of aberrant innate immune activation in AGS Here, we establish that, contrary to expectations, RNase H2 is required for efficient LINE-1 retrotransposition. As RNase H1 overexpression partially rescues the defect in RNase H2 null cells, we propose a model in which RNase H2 degrades the LINE-1 RNA after reverse transcription, allowing retrotransposition to be completed. This also explains how LINE-1 elements can retrotranspose efficiently without their own RNase H activity. Our findings appear to be at odds with LINE-1-derived nucleic acids driving autoinflammation in AGS.


Subject(s)
Autoimmune Diseases of the Nervous System/genetics , Long Interspersed Nucleotide Elements/genetics , Nervous System Malformations/genetics , Ribonuclease H/genetics , Cell Line, Tumor , Gene Knockout Techniques , HCT116 Cells , HeLa Cells , Humans , Reverse Transcription/genetics , Ribonuclease H/biosynthesis
2.
Methods Mol Biol ; 941: 9-18, 2012.
Article in English | MEDLINE | ID: mdl-23065550

ABSTRACT

In vivo overproduction of tRNA chimeras yields an RNA insert within a tRNA scaffold. For some applications, it may be necessary to discard the scaffold. Here we present a protocol for selective cleavage of the RNA of interest from the tRNA scaffold, using RNase H and two DNA oligonucleotides. After cleavage, we show that the RNA of interest can be isolated in a one-step purification. This method has, in particular, applications in structural investigations of RNA.


Subject(s)
RNA Cleavage , RNA, Ribosomal, 16S/metabolism , RNA, Transfer/metabolism , Ribonuclease H/metabolism , Electrophoresis, Polyacrylamide Gel , RNA, Ribosomal, 16S/isolation & purification , Ribonuclease H/biosynthesis , Ribonuclease H/isolation & purification , Staining and Labeling
3.
Protein Expr Purif ; 55(1): 93-9, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17507239

ABSTRACT

The RNase H domain of human hepatitis B virus (HBV) polymerase is an attractive molecular target for the development of new anti-HBV drugs. In this study, a synthetic gene coding for HBV RNase H was assembled from 12 oligonucleotides and expressed in Escherichia coli. The encoded protein was then recovered from inclusion bodies, purified, and refolded by a dilution-dialysis procedure in the presence of a low concentration of lauroylsarcosine (0.01%). The presence of the detergent was an absolute requirement for solubility, suggesting that the untagged RNase H might have exposed hydrophobic regions that need to be shielded from the solvent. The structural identity of the protein was confirmed by N-terminal amino acid sequence analysis and mass spectrometry. The enzymatic activity of HBV RNase H was then tested by a recently developed fluorometric assay and was found to be only slightly lower than that registered with the entire HIV-1 reverse transcriptase. Finally, a structural model of the enzyme showed that H715, R744 and K745 may be involved in substrate recognition.


Subject(s)
Genes, Synthetic , Hepatitis B virus/enzymology , Ribonuclease H/biosynthesis , Viral Nonstructural Proteins/biosynthesis , Amino Acid Sequence , Escherichia coli/genetics , Genome, Viral , Humans , Mass Spectrometry , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary , Ribonuclease H/chemistry , Ribonuclease H/genetics , Substrate Specificity , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
4.
J Biotechnol ; 122(2): 186-97, 2006 Mar 23.
Article in English | MEDLINE | ID: mdl-16233926

ABSTRACT

The progressive cerebral deposition of a 40-42 residues amyloid beta-peptide (Abeta) is regarded as a major factor in the onset of the Alzheimer's disease. It has recently been shown that Abeta(1-40) is cleaved by Escherichia coli pitrilysin, a homologue of insulysin, at a specific site. To facilitate the studies on a recognition mechanism of Abeta by pitrilysin, an overproduction system of Abeta(1-40) as a fusion protein with E. coli RNase HI was constructed. This fusion protein was designed such that an Abeta(1-40) derivative, Abeta(1-40)*, in which Lys16 and Lys28 of Abeta(1-40) are simultaneously replaced by Ala, is attached to the C-terminus of E. coli RNase HI and Abeta(1-40)* is separated from RNase HI upon cleavage with lysyl endopeptidase. The fusion protein was overproduced in E. coli in inclusion bodies, solubilized and purified in the presence of guanidine hydrochloride, and cleaved by lysyl endopeptidase. Abeta(1-40)* was purified from the resultant peptide fragments by reverse-phase HPLC. Measurement of the far-UV CD spectra suggests that Abeta(1-40)* is conformationally similar to Abeta(1-40). However, the thioflavin T binding assay suggests that Abeta(1-40)* is more amyloidogenic than Abeta(1-40). Nevertheless, Abeta(1-40)* was cleaved by pitrilysin at the site identical to that in Abeta(1-40).


Subject(s)
Amyloid beta-Peptides/chemistry , Escherichia coli/metabolism , Metalloendopeptidases/chemistry , Peptide Fragments/chemistry , Protein Engineering , Recombinant Fusion Proteins/chemistry , Ribonuclease H/chemistry , Amino Acid Sequence , Amyloid beta-Peptides/biosynthesis , Amyloid beta-Peptides/isolation & purification , Escherichia coli/genetics , Intranuclear Inclusion Bodies/metabolism , Molecular Sequence Data , Peptide Fragments/biosynthesis , Peptide Fragments/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Ribonuclease H/biosynthesis , Ribonuclease H/isolation & purification
5.
Biochem J ; 381(Pt 3): 795-802, 2004 Aug 01.
Article in English | MEDLINE | ID: mdl-15115438

ABSTRACT

All the archaeal genomes sequenced to date contain a single Type 2 RNase H gene. We found that the genome of a halophilic archaeon, Halobacterium sp. NRC-1, contains an open reading frame with similarity to Type 1 RNase H. The protein encoded by the Vng0255c gene, possessed amino acid sequence identities of 33% with Escherichia coli RNase HI and 34% with a Bacillus subtilis RNase HI homologue. The B. subtilis RNase HI homologue, however, lacks amino acid sequences corresponding to a basic protrusion region of the E. coli RNase HI, and the Vng0255c has the similar deletion. As this deletion apparently conferred a complete loss of RNase H activity on the B. subtilis RNase HI homologue protein, the Vng0255c product was expected to exhibit no RNase H activity. However, the purified recombinant Vng0255c protein specifically cleaved an RNA strand of the RNA/DNA hybrid in vitro, and when the Vng0255c gene was expressed in an E. coli strain MIC2067 it could suppress the temperature-sensitive growth defect associated with the loss of RNase H enzymes of this strain. These results in vitro and in vivo strongly indicate that the Halobacterium Vng0255c is the first archaeal Type 1 RNase H. This enzyme, unlike other Type 1 RNases H, was able to cleave an Okazaki fragment-like substrate at the junction between the 3'-side of ribonucleotide and 5'-side of deoxyribonucleotide. It is likely that the archaeal Type 1 RNase H plays a role in the removal of the last ribonucleotide of the RNA primer from the Okazaki fragment during DNA replication.


Subject(s)
Halobacterium/enzymology , Halobacterium/genetics , Ribonuclease H/genetics , Amino Acid Sequence/genetics , Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Cations, Divalent/chemistry , Cloning, Molecular/methods , DNA/chemistry , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/metabolism , Evolution, Molecular , Genetic Complementation Test/methods , Genome, Archaeal , Molecular Sequence Data , Nucleic Acid Heteroduplexes/chemistry , RNA/chemistry , RNA/metabolism , Ribonuclease H/biosynthesis , Ribonuclease H/chemistry , Ribonuclease H/metabolism , Sequence Homology, Amino Acid , Substrate Specificity/genetics
6.
Biochem Biophys Res Commun ; 317(2): 321-9, 2004 Apr 30.
Article in English | MEDLINE | ID: mdl-15063760

ABSTRACT

This report describes a procedure to generate enzymatically active, isolated HIV RNase H domain. In contrast to previously described preparations, the RNA cleavage activity of the untagged RNase H domain was surprisingly similar to that of the full-length HIV-RT protein. Signature cleavages at 18 and 9 nucleotides downstream of a recessed RNA 5'-end were retained with the isolated RNase H domain. Activity was strongly decreased by deletion of 3 amino acids from the C-terminus, consistent with an important structural or functional role of the C-terminal alpha-helix. A prototype N-hydroxyimide (2-hydroxy-4H-isoquinoline-1,3-dione) was found to inhibit the activity of the isolated HIV RNase H domain as well as the RNase H activity of full-length HIV reverse transcriptase. In contrast, the compound did not significantly inhibit the structurally closely related Escherichia coli RNase HI. Specific binding of N-hydroxyimide compounds to the isolated RNase H domain was observed by protein fluorescence quenching.


Subject(s)
HIV Reverse Transcriptase/chemistry , HIV-1/enzymology , Imides/chemistry , Ribonuclease H/chemistry , Amino Acid Substitution , Binding Sites , Enzyme Activation , Enzyme Inhibitors/chemistry , HIV Reverse Transcriptase/biosynthesis , HIV Reverse Transcriptase/genetics , HIV-1/genetics , Hydrolysis , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Ribonuclease H/biosynthesis , Ribonuclease H/genetics , Ribonuclease H/isolation & purification , Sensitivity and Specificity , Structure-Activity Relationship , Substrate Specificity
7.
FEMS Microbiol Lett ; 221(2): 237-42, 2003 Apr 25.
Article in English | MEDLINE | ID: mdl-12725933

ABSTRACT

Induction of stress proteins in response to heat shock was found to be reduced significantly in Escherichia coli with DeltatopA mutation. RNase H overexpression in the DeltatopA mutant partially restored the sigma(32)-dependent induction of stress genes in response to high temperature and ethanol. The presence of overexpressed RNase H also improved the survival rate of the DeltatopA mutant after high temperature and oxidative challenges. Topoisomerase I is likely required during stress response for preventing accumulation of transcription-driven hypernegative supercoiling and R-loop formation at induced stress genes loci.


Subject(s)
DNA Topoisomerases, Type I/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Heat-Shock Proteins/biosynthesis , Ribonuclease H/biosynthesis , DNA, Bacterial/analysis , Escherichia coli/enzymology , Escherichia coli/growth & development , Mutation , Nucleic Acid Conformation , Temperature , Transcription, Genetic
8.
J Appl Physiol (1985) ; 95(3): 1201-6, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12766179

ABSTRACT

Changes in gene expression during recovery from high-intensity, intermittent, one-legged exercise were studied before and after 5.5 wk of training. Genes related to metabolism, as well as Na+, K+, and pH homeostasis, were selected for analyses. After the same work was performed before and after the training period, several muscle biopsies were obtained from vastus lateralis muscle. In the untrained state, the Na+-K+-ATPase alpha1-subunit mRNA level was approximately threefold higher (P < 0.01) at 0, 1, and 3 h after exercise, relative to the preexercise resting level. After 3-5 h of recovery in the untrained state, pyruvate dehydrogenase kinase 4 and hexokinase II mRNA levels were elevated 13-fold (P < 0.001) and 6-fold (P < 0.01), respectively. However, after the training period, only pyruvate dehydrogenase kinase 4 mRNA levels were elevated (P < 0.05) during the recovery period. No changes in resting mRNA levels were observed as a result of training. In conclusion, cellular adaptations to high-intensity exercise training may, in part, be induced by transcriptional regulation. After training, the transcriptional response to an exercise bout at a given workload is diminished.


Subject(s)
Exercise/physiology , Gene Expression Regulation/physiology , Homeostasis/genetics , Physical Fitness/physiology , ATP-Binding Cassette Transporters , Adult , DNA Primers , Gene Expression Regulation/genetics , Humans , Ions/metabolism , KATP Channels , Male , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiology , Phosphofructokinases/biosynthesis , Phosphofructokinases/genetics , Potassium Channels/biosynthesis , Potassium Channels/genetics , Potassium Channels, Inwardly Rectifying , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Ribonuclease H/biosynthesis , Ribonuclease H/genetics , Sodium-Potassium-Exchanging ATPase/metabolism
9.
Biochem Pharmacol ; 61(4): 467-76, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11226381

ABSTRACT

Ribonuclease H (RNase H), an enzyme that cleaves an RNA sequence base-paired with a complementary DNA sequence, is proposed to be the mediator of antisense phosphorothioate oligonucleotide (S-oligo) lethality in a cell. To understand the role of RNase H in the killing of the parasitic protozoan Leishmania by antisense S-oligos, we expressed an episomal copy of the Trypanosoma brucei RNase H1 gene inside L. amazonensis promastigotes and amastigotes that constitutively express firefly luciferase. Our hypothesis was that S-oligo-directed degradation of target mRNA is facilitated in a cell that has higher RNase H activity. Increased inhibition of luciferase mRNA expression by anti-luciferase S-oligo and by anti-miniexon S-oligo in these stably transfected promastigotes overexpressing RNase H1 was correlated to the higher activity of RNase H in these cells. The efficiency of killing of the RNase H overexpressing amastigotes inside L. amazonensis-infected macrophages by anti-miniexon S-oligo was higher than in the control cells. Thus, RNase H appears to play an important role in the antisense S-oligo-mediated killing of Leishmania. Chemical modification of S-oligos that stimulate RNase H and/or co-treatment of cells with an activator of RNase H may be useful for developing an antisense approach against leishmaniasis. The transgenic Leishmania cells overexpressing RNase H should be a good model system for the antisense-mediated gene expression ablation studies in these parasites.


Subject(s)
Antiprotozoal Agents/pharmacology , Leishmania/drug effects , Oligonucleotides, Antisense/pharmacology , Ribonuclease H/physiology , Thionucleosides/pharmacology , Animals , Base Sequence , DNA, Protozoan/analysis , Drug Evaluation, Preclinical , Gene Expression/drug effects , Leishmania/enzymology , Leishmania/genetics , Leishmania/metabolism , Luciferases/biosynthesis , Luciferases/genetics , Molecular Sequence Data , RNA, Messenger/biosynthesis , RNA, Messenger/drug effects , Ribonuclease H/biosynthesis , Transfection , Trypanosoma brucei brucei/drug effects , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/metabolism
10.
Pharmazie ; 55(8): 615-7, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10989842

ABSTRACT

Two major hurdles to the widespread use of synthetic nucleic acids as drugs are the biological stability of the compounds and efficiency of cellular penetration. Recent advances in the chemistry of nucleic acids has given rise to highly stable derivatives with an anhydrohexitol backbone. This report addresses the cellular uptake of these molecules. We show that the uptake of HNA in the absence of a carrier is very low but HNA is efficiently internalized with a range of transfection reagents.


Subject(s)
Hexosephosphates/metabolism , Nucleic Acids/metabolism , Oligonucleotides, Antisense/metabolism , Fluorescent Dyes , HeLa Cells , Humans , Ribonuclease H/biosynthesis
11.
J Biochem ; 127(5): 895-9, 2000 May.
Article in English | MEDLINE | ID: mdl-10788800

ABSTRACT

Escherichia coli RNase HII is composed of 198 amino acid residues. The enzyme has been overproduced in an insoluble form, purified in a urea-denatured form, and refolded with poor yield [M. Itaya (1990) Proc. Natl. Acad. Sci. USA 87, 8587-8591]. To facilitate the preparation of the enzyme in an amount sufficient for physicochemical studies, we constructed an overproducing strain in which E. coli RNase HII is produced in a soluble form. The enzyme was purified from this strain and its biochemical and physicochemical properties were characterized. The good agreement in the molecular weights estimated from SDS-PAGE (23,000) and gel filtration (22,000) suggests that the enzyme acts as a monomer. From the far-UV circular dichroism spectrum, its helical content was calculated to be 23%. The enzyme showed Mn(2+)-dependent RNase H activity. Its specific activity determined using (3)H-labeled M13 RNA/DNA hybrid as a substrate was comparable to but slightly higher than that of the refolded enzyme, indicating that the enzyme overproduced and purified in a soluble form is more suitable for structural and functional analyses than the refolded enzyme.


Subject(s)
Bacterial Proteins/biosynthesis , Escherichia coli/enzymology , Recombinant Proteins/biosynthesis , Ribonuclease H/biosynthesis , Bacterial Proteins/genetics , DNA, Viral/metabolism , Escherichia coli/genetics , Nucleic Acid Heteroduplexes/metabolism , RNA, Viral/metabolism , Ribonuclease H/genetics , Ribonuclease H/metabolism , Sequence Analysis, Protein , Solubility
13.
Biochem Cell Biol ; 77(2): 119-26, 1999.
Article in English | MEDLINE | ID: mdl-10438146

ABSTRACT

Genome replication of hepadnavirus proceeds by reverse transcription from a viral pregenomic RNA template by a virally encoded polymerase that possesses protein-priming, reverse transcriptase, DNA polymerase, and RNase H activities. Characterization of this enzyme has been hampered by failure to purify an active enzyme from virions and difficulties in expressing an active polymerase in heterologous systems. In this study, we constructed human hepatitis B virus polymerase cDNA under the control of a phage T7 promoter and expressed it in a rabbit reticulocyte lysate-coupled transcription-translation system. In vitro site-directed mutagenesis confirmed that the recombinant polymerase cDNA produced three products: a full-length protein (approximately 94 kDa), an internally initiated protein (approximately 81 kDa), and an N-terminal protein (approximately 40 kDa). The in vitro expressed polymerase possessed protein priming activity, as demonstrated by 32P-dGTP-labeling of the full size polymerase and the N-terminal portion of the molecule in an in vitro priming assay. The polymerase also exhibited polymerization activity, as detected in an in vitro polymerase assay by incorporation of radionucleotides into acid-precipitable polynucleotides and by synthesis of human hepatitis B virus (HBV) specific DNA with product lengths between 100 and 500 nucleotides. In addition, the polymerase was found to use an RNA sequence bearing HBV DR1/epsilon stem-loop motif as a template for DNA synthesis. Both the protein-priming and the reverse transcription activities of this recombinant polymerase are dependent on the RNA fragment containing the HBV DR1/epsilon stem-loop sequence known to be required for the polymerase activities. The in vitro systems used in this study will be applicable to further functional and biochemical studies of this enzyme.


Subject(s)
DNA-Directed DNA Polymerase/biosynthesis , Hepatitis B virus/enzymology , Protein Biosynthesis , Transcription, Genetic , Animals , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Deoxyguanine Nucleotides , Humans , Phosphorus Radioisotopes , RNA-Directed DNA Polymerase/biosynthesis , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/metabolism , Rabbits , Recombination, Genetic , Ribonuclease H/biosynthesis , Ribonuclease H/genetics , Ribonuclease H/metabolism , Templates, Genetic
14.
Proc Natl Acad Sci U S A ; 95(22): 12872-7, 1998 Oct 27.
Article in English | MEDLINE | ID: mdl-9789007

ABSTRACT

Two RNases H of mammalian tissues have been described: RNase HI, the activity of which was found to rise during DNA replication, and RNase HII, which may be involved in transcription. RNase HI is the major mammalian enzyme representing around 85% of the total RNase H activity in the cell. By using highly purified calf thymus RNase HI we identified the sequences of several tryptic peptides. This information enabled us to determine the sequence of the cDNA coding for the large subunit of human RNase HI. The corresponding ORF of 897 nt defines a polypeptide of relative molecular mass of 33,367, which is in agreement with the molecular mass obtained earlier by SDS/PAGE. Expression of the cloned ORF in Escherichia coli leads to a polypeptide, which is specifically recognized by an antiserum raised against calf thymus RNase HI. Interestingly, the deduced amino acid sequence of this subunit of human RNase HI displays significant homology to RNase HII from E. coli, an enzyme of unknown function and previously judged as a minor activity. This finding suggests an evolutionary link between the mammalian RNases HI and the prokaryotic RNases HII. The idea of a mammalian RNase HI large subunit being a strongly conserved protein is substantiated by the existence of homologous ORFs in the genomes of other eukaryotes and of all eubacteria and archaebacteria that have been completely sequenced.


Subject(s)
Ribonuclease H/biosynthesis , Ribonuclease H/genetics , Amino Acid Sequence , Animals , Bacteria/enzymology , Base Sequence , Cattle , Cloning, Molecular , DNA, Complementary , Humans , Molecular Sequence Data , Open Reading Frames , Peptide Fragments/chemistry , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Ribonuclease H/chemistry , Sequence Alignment , Sequence Homology, Amino Acid , Thymus Gland/enzymology , Trypsin
15.
Biochemistry ; 37(20): 7478-86, 1998 May 19.
Article in English | MEDLINE | ID: mdl-9585561

ABSTRACT

In recent publications, oligonucleotides joined by 2',5'-linkages were found to bind to complementary single-stranded RNA but to bind weakly, or not at all, to single-stranded DNA [e.g., P. A. Giannaris and M. J. Damha (1993) Nucleic Acids Res. 21, 4742-4749]. In this work, the biochemical and physicochemical properties of 2',5'-linked oligoribonucleotides containing mixed sequences of the four nucleobases (A, G, C, and U) were evaluated. CD spectra of RNA:2', 5'-RNA duplexes were compared with the spectra of DNA:DNA, RNA:RNA, and DNA:RNA duplexes of the same base sequence. The CD results indicated that the RNA:2',5'-RNA duplex structure more closely resembles the structure of the RNA:DNA hybrid, being more A-form than B-form in character. The melting temperature (Tm) values of the backbone-modified duplexes were compared with the Tm values of the unmodified duplexes. The order of thermal stability was RNA:RNA > DNA:DNA approximately RNA:DNA approximately DNA:RNA > RNA:2',5'-RNA > 2',5'-RNA:2',5'-RNA >> DNA:2',5'-RNA (undetected). RNA:2',5'-RNA duplexes are not substrates of the enzyme RNase H (Escherichia coli, or HIV-1 reverse transcriptase), but they can inhibit the RNase H-mediated cleavage of a natural DNA:RNA substrate. Structural models that are consistent with the selective association properties of 2',5'-linked oligonucleotides are discussed.


Subject(s)
Nucleic Acid Heteroduplexes/chemistry , Oligoribonucleotides/chemistry , RNA/chemistry , Carbohydrate Conformation , Chemical Phenomena , Chemistry, Physical , Circular Dichroism , DNA/chemistry , Enzyme Induction/drug effects , Hot Temperature , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes/chemical synthesis , Nucleic Acid Heteroduplexes/pharmacology , Oligoribonucleotides/chemical synthesis , Oligoribonucleotides/pharmacology , RNA/chemical synthesis , RNA/pharmacology , Ribonuclease H/biosynthesis , Ribonuclease H/metabolism , Spectrophotometry, Ultraviolet , Substrate Specificity/drug effects , Substrate Specificity/genetics
16.
J Biol Chem ; 273(16): 9785-9, 1998 Apr 17.
Article in English | MEDLINE | ID: mdl-9545316

ABSTRACT

The existence of retroviral reverse transcriptases as monomers or dimers is rather intriguing. A classical example of the former is murine leukemia virus reverse transcriptase (MuLV RT), while human immunodeficiency virus type 1 (HIV-1) RT represents the latter. A careful scrutiny of the amino acid sequence alignment of the two enzymes pinpoints the region tentatively responsible for this phenomenon. We report here the construction of a chimeric enzyme containing the first 425 amino acid residues from the N-terminal domain of HIV-1 RT and 200 amino acid residues from the C-terminal domain of MuLV RT. The chimeric enzyme exists as a monomer with intact DNA polymerase and RNase-H functions.


Subject(s)
HIV Reverse Transcriptase/metabolism , RNA-Directed DNA Polymerase/metabolism , Ribonuclease H/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , HIV Reverse Transcriptase/biosynthesis , HIV Reverse Transcriptase/chemistry , HIV-1/enzymology , Humans , Mice , Molecular Sequence Data , Moloney murine leukemia virus/enzymology , Polymerase Chain Reaction , RNA-Directed DNA Polymerase/biosynthesis , RNA-Directed DNA Polymerase/chemistry , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Ribonuclease H/biosynthesis , Ribonuclease H/chemistry , Sequence Alignment , Sequence Homology, Amino Acid
17.
Antisense Nucleic Acid Drug Dev ; 8(1): 53-61, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9512096

ABSTRACT

We have cloned, expressed, and purified to electrophoretic homogeneity a human RNase H. The enzyme has a molecular weight of 32 kDa, is Mg2+ dependent, and is inhibited by Mn2+ and N-ethylmaleimide. Its molecular weight and cleavage characteristics are consistent with type 2 human RNase H. The human RNase H we have cloned is highly homologous to Escherichia coli RNase HI (33.6% amino acid identity) and to other RNase H enzymes homologous to E. coli RNase HI. The enzyme is encoded by a single gene that is at least 10 kb in length and is expressed ubiquitously in human cells and tissues.


Subject(s)
Ribonuclease H/genetics , Amino Acid Sequence , Cloning, Molecular , DNA, Complementary/genetics , Escherichia coli/genetics , Gene Library , Humans , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Ribonuclease H/biosynthesis , Ribonuclease H/classification , Sequence Analysis, DNA , Sequence Homology, Amino Acid
18.
FEBS Lett ; 402(2-3): 177-80, 1997 Feb 03.
Article in English | MEDLINE | ID: mdl-9037190

ABSTRACT

Random sequences of 120-130 amino acid residues were inserted into a surface loop region of Escherichia coli RNase HI. This library was screened and about 10% of the clones were found to retain RNase H activity. Subsequent random mutagenesis led to an increase in RNase H activity and solubility of the protein. The inserted regions were found not to contribute to the secondary structure of the mutant protein. The high frequency of insertion of flexible sequences and the increase in the protein's function by further mutagenesis simulate one of the events in protein evolution.


Subject(s)
Escherichia coli/enzymology , Mutagenesis, Insertional , Protein Structure, Secondary , Ribonuclease H/chemistry , Ribonuclease H/metabolism , Amino Acid Sequence , Circular Dichroism , Cloning, Molecular , Gene Library , Models, Structural , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribonuclease H/biosynthesis
19.
J Virol ; 70(9): 6455-8, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8709281

ABSTRACT

While the intracellular pool of encapsidated hepatitis B viral DNA contains genomes in all stages of DNA replication, serum-derived virions contain predominantly mature, partially duplex, circular DNA genomes. To account for this finding, Summers and Mason proposed in 1982 that virion envelopment is somehow linked to the state of genomic maturation (J. Summers and W.S. Mason, Cell 29:403-415, 1982). Core gene mutations with phenotypes consistent with this concept have previously been identified in the duck hepatitis B virus (DHBV). Here we show that DHBV polymerase mutants with altered DNA synthesis also display defects in envelopment, and we provide quantitative estimates of the magnitude of the preference for the envelopment of mature DNA. In cells transfected with wild-type DHBV DNA, immature minus-strand DNA represents 18% of the intracellular pool but only 4% of extracellular virion DNA. A point mutation in the C-terminal domain of the polymerase strongly and selectively impairs plus-strand synthesis; in this mutant, the ratio of immature to mature DNA in the intracellular pool rises to 6:1 but is reduced to 1.5:1 in released virions. A missense mutation in the polymerase active site inactivates all viral DNA synthesis but still allows efficient RNA encapsidation; in this mutant, no detectable viral nucleic acid is enveloped and released. Thus, viral DNA synthesis is absolutely required for envelopment and export, and a strong further bias exists in favor of the export of genomes that have completed minus-strand synthesis and at least initiated plus-strand synthesis. These results imply that events within the interior of the nucleocapsid can powerfully influence its interactions with external viral envelope glycoproteins.


Subject(s)
Capsid/biosynthesis , DNA Replication , DNA, Viral/biosynthesis , DNA-Directed DNA Polymerase/genetics , Gene Products, env/metabolism , Hepatitis B Virus, Duck/physiology , Point Mutation , RNA-Directed DNA Polymerase/genetics , Viral Core Proteins/biosynthesis , Virion/physiology , Amino Acid Sequence , Animals , Carcinoma, Hepatocellular , Cell Line , Cells, Cultured , Chickens , DNA-Directed DNA Polymerase/biosynthesis , DNA-Directed DNA Polymerase/metabolism , Ducks , Genome, Viral , Hepatitis B Virus, Duck/genetics , Liver/cytology , Liver/metabolism , Liver Neoplasms , Molecular Sequence Data , RNA, Viral/metabolism , RNA-Directed DNA Polymerase/metabolism , Recombinant Proteins/metabolism , Ribonuclease H/biosynthesis , Ribonuclease H/genetics , Transfection , Tumor Cells, Cultured , Virus Replication
20.
Gene ; 165(1): 71-5, 1995 Nov 07.
Article in English | MEDLINE | ID: mdl-7489919

ABSTRACT

Activity gel analysis of cell extracts from slow- and fast-growing mycobacteria confirmed the presence of several RNase H activities in both classes of organism. The rnhA gene from Mycobacterium smegmatis (Ms) was subsequently cloned using an internal gene segment probe [Mizrahi et al., Gene 136 (1993) 287-290]. The gene encodes a polypeptide of 159 amino acids that shares 50% identity with the RNase HI from Escherichia coli (Ec). However, unlike its counterparts from Gram- bacteria, Ms rnhA does not form an overlapping divergent transcriptional unit with dnaQ (encoding the epsilon (proofreading) subunit of DNA polymerase III). Ms RNase HI was overproduced in Ec as an enzymatically active maltose-binding protein (MBP) fusion protein which cleaved the RNA strand of an RNA.DNA hybrid with a similar site selectivity to that of its Ec homologue.


Subject(s)
Escherichia coli/genetics , Mycobacterium/enzymology , Ribonuclease H/genetics , Base Sequence , Cloning, Molecular , Enzyme Activation , Escherichia coli/enzymology , Molecular Sequence Data , Mycobacterium/genetics , Ribonuclease H/biosynthesis , Ribonuclease H/metabolism , Sequence Analysis
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