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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutations in the primer grip region of human immunodeficiency virus reverse transcriptase (HIV-RT) affect its replication fidelity. The primer grip region (residues 227-235) correctly positions the 3'-ends of primers. Point mutations were created by alanine substitution at positions 224-235. Error frequencies were measured by extension of a dG:dA primer-template mismatch. Mutants E224A, P225A, P226A, L228A, and E233A were approximately equal to the wild type in their ability to extend the mismatch. Mutants F227A, W229A, M230A, G231A, and Y232A extended 40, 66, 54, 72, and 76% less efficiently past a dG:dA mismatch compared with the wild type. We also examined the misinsertion rates of dG, dC, or dA across from a DNA template dA using RT mutants F227A and W229A. Mutant W229A exhibited high fidelity and did not produce a dG:dA or dC:dA mismatch. Interestingly, mutant F227A displayed high fidelity for dG:dA and dC:dA mismatches but low fidelity for dA:dA misinsertions. This indicates that F227A discriminates against particular base substitutions. However, a primer extension assay with three dNTPs showed that F227A generally displays higher fidelity than the wild type RT. Clearly, primer grip mutations can improve or worsen either the overall or base-specific fidelity of HIV-RT. We hypothesize that wild type RT has evolved to a fidelity that allows genetic variation without compromising yield of viable viruses.
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PMID:Mutations in the primer grip region of HIV reverse transcriptase can increase replication fidelity. 1049 70

The TCR structure of T cell hybridomas recognizing a tumor glycan-defined epitope has been studied using reverse transcriptase-PCR and gene sequencing. The hybridomas had been raised against a glycopeptide, T72(Tn), consisting of the mouse hemoglobin-derived decapeptide Hb(67 - 76), O-glycoslated in position 72 with alpha-D-GalNAc. The glycan-specific hybridomas varied widely in their use of Valpha genes although Valpha4 was predominant, being present in one third of them. The Vbeta gene usage was more restricted and dominated by Vbeta1 and Vbeta15. There was no correlation between Valpha and Vbeta usage and antigen fine specificity of the hybridomas. The overall amino acid composition of the complementarity-determining region (CDR) 3 of the hybridomas was dominated by small polar residues such as Gly, Asn, Ser, Glu and Ala, amino acids reported in the literature to be frequent in glycan-recognizing proteins. Furthermore, the CDR3 of most hybridomas also contained an aromatic residue with preference for Tyr. A few of the hybridomas raised against the T72(Tn) glycopeptide were peptide specific, i. e. they responded to the unglycosylated peptide only. The amino acid usage of their CDR3 regions was not radically different from that of the glycopeptide specific hybridomas. They also preferentially used Valpha4. However, Vbeta4 and Vbeta8 were the dominating beta chains.
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PMID:Shared structural motifs in TCR of glycopeptide-recognizing T cell hybridomas. 1050 50

Arginine 72 in human immunodeficiency virus type 1 reverse transcriptase (RT), a highly conserved residue among retroviral polymerases and telomerases, forms part of the binding pocket for the nascent base pair. We show here that replacement of Arg(72) by alanine strongly alters fidelity in a highly unusual manner. R72A reverse transcriptase is a frameshift and base substitution antimutator polymerase whose increased fidelity results both from increased nucleotide selectivity and from a decreased ability to extend mismatched primer termini. Thus, Arg(72)-substrate interactions in wild-type human immunodeficiency virus type 1 RT can stabilize incorrect nucleotides allowing misinsertion and promoting extension of mismatched and perhaps misaligned template-primers. In contrast to the higher fidelity at most sites, R72A RT is highly error-prone for misincorporations opposite template T in the sequence context: 5'-CTGG. Surprisingly, this results mostly from a 1200-fold increase in the apparent K(m) for correct dAMP incorporation. Thus, Arg(72) interactions with substrate are critical for the stability of the correct T.dAMP base pair when the 5'-CTGG sequence is present in the binding pocket for the nascent base pair. Collectively, the data show that a mutant polymerase may yield higher than normal average replication fidelity, yet paradoxically place specific sequences at very high risk of mutation.
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PMID:Uniquely altered DNA replication fidelity conferred by an amino acid change in the nucleotide binding pocket of human immunodeficiency virus type 1 reverse transcriptase. 1055 58

Telomerase is a ribonucleoprotein complex that adds telomeric DNA repeats to the ends of most eukaryotic chromosomes. The reverse transcriptase subunit of telomerase (TERT) differs from retroviral reverse transcriptases in having a long basic amino-terminal extension. We made a large library containing random mutations in the amino terminus of the EST2 gene, which encodes the Saccharomyces cerevisiae TERT, and selected functional alleles by their ability to rescue senescence of telomerase-negative cells. Through analysis of 265 mutations, the amino terminus of Est2p was found to contain at least four essential regions. This domain structure was verified by a combination of deletion and alanine-block mutations. Mutations within two essential domains of the protein reduced RNA binding, suggesting that the amino terminus of Est2p makes important contacts with the intrinsic RNA component of telomerase. A mutant close to the amino terminus retained RNA binding and in vitro enzymatic activity but was defective in vivo, suggesting a role in interaction with other macromolecular components of telomerase.
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PMID:Essential functions of amino-terminal domains in the yeast telomerase catalytic subunit revealed by selection for viable mutants. 1055 13

Deoxyribonuclease I (DNase I) was purified from the hen pancreas to electrophoretic homogeneity using six-step column chromatography. The purified enzyme showed a molecular mass of about 33 kDa and maximum activity at pH 7.0. It required divalent cations, Mg2+ and Ca2+, for its activity and was inhibited by EDTA, EGTA and an antibody specific to the purified enzyme but not by G-actin. A 1066-bp cDNA encoding hen DNase I was constructed from the total RNA of a hen pancreas using a combination of the reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends methods, followed by sequencing. The cDNA was expressed in Escherichia coli, and the recombinant polypeptide exhibited significant enzyme activity. The mature hen DNase I protein was found to consist of 262 amino acids. In human and bovine DNase I four amino acid residues, Glu-13, Tyr-65, Val-67 and Ala-114 are involved in actin binding, whereas in the hen DNase I these positions were occupied by Asp, Phe, Ser and Phe, respectively. A survey of the DNase I distribution in 15 hen tissues showed that the pancreas had the highest levels of both DNase I enzyme activity and DNase I gene expression. The results of our phylogenetic and immunological analyses indicate that the hen DNase I is not closely related to the mammalian enzymes. This is the first report in which has been described the results of molecular, biochemical and immunological analyses on hen DNase I.
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PMID:Molecular, biochemical and immunological studies of hen pancreatic deoxyribonuclease I. 1060 24

Glycolipid transfer protein (GLTP) is a small (23-24 kDa), basic protein (pI congruent with 9.0) that accelerates the intermembrane transfer of various glycolipids. Here, we report the first cloning of cDNAs that encode the bovine and porcine GLTPs. The cDNA open reading frame for bovine GLTP was constructed by bridge-overlapping extension polymerase chain reaction (PCR) after obtaining partial coding cDNA clones by hot start, seminested, and rapid amplification of cDNA ends-PCR. The cDNA open reading frame for porcine GLTP was constructed by reverse transcriptase-PCR. The encoded amino acid sequences in the full-length bovine and porcine cDNAs were identical, consisting of 209 amino acid residues, and were nearly the same as the published sequence determined by Edman degradation. The cDNA encoded one additional amino acid at the N terminus (methionine), arginine at positions 10 and 200 instead of lysine, and threonine at position 65 instead of alanine. Expression of GLTP-cDNA in Escherichia coli using pGEX-6P-1 vector resulted in glutathione S-transferase (GST)-GLTP fusion protein. Regulation of growth and induction conditions led to approximately 50% of expressed fusion protein being soluble and active. Proteolytic cleavage of GST-GLTP fusion protein (bound to GST-Sepharose) and affinity purification resulted in fully active GLTP. Northern blot analyses of bovine tissues showed a single transcript of approximately 2.2 kilobases and the following hierarchy of mRNA levels: cerebrum > kidney > spleen congruent with lung congruent with cerebellum > liver > heart muscle. Reverse transcriptase-PCR analyses of mRNA levels supported the Northern blot results.
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PMID:Cloning and expression of glycolipid transfer protein from bovine and porcine brain. 1067 54

We describe a new human immunodeficiency virus type 1 (HIV-1) mutational pattern associated with phenotypic resistance to lamivudine (3TC) in the absence of the characteristic replacement of methionine by valine at position 184 (M184V) of reverse transcriptase. Combined genotypic and phenotypic analyses of clinical isolates revealed the presence of moderate levels of phenotypic resistance (between 4- and 50-fold) to 3TC in a subset of isolates that did not harbor the M184V mutation. Mutational cluster analysis and comparison with the phenotypic data revealed a significant correlation between moderate phenotypic 3TC resistance and an increased incidence of replacement of glutamic acid by aspartic acid or alanine and of valine by isoleucine at residues 44 and 118 of reverse transcriptase, respectively. This occurred predominantly in those isolates harboring zidovudine resistance-associated mutations (41L, 215Y). The requirement of the combination of mutations 41L and 215Y with mutations 44D and 44A and/or 118I for phenotypic 3TC resistance was confirmed by site-directed mutagenesis experiments. These data support the assumption that HIV-1 may have access to several different genetic pathways to escape drug pressure or that the increase in the frequency of particular mutations may affect susceptibility to drugs that have never been part of a particular regimen.
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PMID:A novel human immunodeficiency virus type 1 reverse transcriptase mutational pattern confers phenotypic lamivudine resistance in the absence of mutation 184V. 1068 19

Biochemical and molecular modeling studies of human immunodeficiency virus type 1 reverse transcriptase (RT) have revealed that a structural element, the minor groove binding track (MGBT), is important for both replication frameshift fidelity and processivity. The MGBT interactions occur in the DNA minor groove from the second through sixth base pair from the primer 3'-terminus where the DNA undergoes a structural transition from A-like to B-form DNA. Alanine-scanning mutagenesis had previously demonstrated that Gly(262) and Trp(266) of the MGBT contributes important DNA interactions. To probe the molecular interactions occurring in this critical region, eight mutants of RT were studied in which alternate residues were substituted for Trp(266). These enzymes were characterized in primer extension assays in which the template DNA was adducted at a single adenine by either R- or S-enantiomers of styrene oxide. These lesions failed to block DNA polymerization by wild-type RT, yet the Trp(266) mutants and an alanine mutant of Gly(262) terminated synthesis on styrene oxide-adducted templates. Significantly, the sites of termination occurred primarily 1 and 3 bases following adduct bypass, when the lesion was positioned in the major groove of the template-primer stem. These results indicate that residue 266 serves as a "protein sensor" of altered minor groove interactions and identifies which base pair interactions are altered by these lesions. In addition, the major groove lesion must alter important structural transitions in the template-primer stem, such as minor groove widening, that allow RT access to the minor groove.
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PMID:Vertical-scanning mutagenesis of a critical tryptophan in the "minor groove binding track" of HIV-1 reverse transcriptase. Major groove DNA adducts identify specific protein interactions in the minor groove. 1074 90

Substitution of particular residues postulated to have a role in active site architecture can alter the overall fidelity of DNA polymerization by HIV-1. The effects of this kind of substitution were determined in a lacZ-based assay using HIV-1 reverse transcriptase with specifically mutated residues. We found that the reported higher fidelity of nucleotide incorporation by the Met184-->Val and Glu89-->Gly mutant reverse transcriptases (RTs) was not reflected in a substantial increase in the overall fidelity for these RT mutants. For the 3TC-resistant Met184-->Val RT mutant an almost wild-type level of overall mutation frequency was observed, while the foscarnet-resistant RTs harbouring the Glu89-->Gly mutation showed only a twofold decrease in mutation frequency. The Tyr183-->Phe mutant RT displayed a slightly lower fidelity than wild-type RT. Conversely, the ddI-resistant RT mutant containing the Leu74-->Val mutation showed a 3.5-fold higher fidelity compared to the wild-type enzyme. Finally, the Tyr115-->Ala substitution rendered the enzyme substantially more error-prone for DNA polymerization. These results correlate with three-dimensional structural studies of the polymerase active site and confirm the postulated impact of the Leu74, Tyr183 and Tyr115 RT residues on the overall fidelity of DNA polymerization by HIV-1 RT.
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PMID:Fidelity analysis of HIV-1 reverse transcriptase mutants with an altered amino-acid sequence at residues Leu74, Glu89, Tyr115, Tyr183 and Met184. 1078 87

The protein catalytic subunit of telomerase (TERT) is a reverse transcriptase (RT) that utilizes an internal RNA molecule as a template for the extension of chromosomal DNA ends. In all retroviral RTs there is a conserved tyrosine two amino acids preceding the catalytic aspartic acids in motif C, a motif that is critical for catalysis. In TERTs, however, this position is a leucine, valine, or phenylalanine. We developed and characterized a robust in vitro reconstitution system for Tetrahymena telomerase and tested the effects of amino acid substitutions on activity. Substitution of the retroviral-like tyrosine in motif C did not change overall enzymatic activity but increased processivity. This increase in processivity correlated with an increased affinity for telomeric DNA primer. Substitution of an alanine did not increase processivity, while substitution of a phenylalanine had an intermediate effect. The data suggest that this amino acid is involved in interactions with the primer in telomerase as in other RTs, and show that mutating an amino acid to that conserved in retroviral RTs makes telomerase more closely resemble these other RTs.
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PMID:A mutant of Tetrahymena telomerase reverse transcriptase with increased processivity. 1080 25


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