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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HIV-1 reverse transcriptase (HIV-1 RT) is a multifunctional enzyme responsible for converting viral RNA into preintegrative DNA during the early stages of viral infection. DNA polymerase and RNase H activities are required, and several conformationally distinct primer-templates must be accommodated by the enzyme during the process. Parameters of interaction between model substrates (ligands) and HIV-1 RT (wild type p66/p51 and the RNase H-deficient mutant p66(E478Q)/p51) (analytes) were estimated by surface plasmon resonance at 25 degrees C, pH 8.0. Binding of RT to the ligands is specific and can be analyzed using a conventional 1:1 binding algorithm. RNA-DNA hybrids with 5'-template overhangs of 6 and 12 nucleotides bind to RT approximately one order of magnitude stronger than the corresponding 36-mer with blunt ends due to slower dissociation. Immobilization of the latter through either the 5'-end of RNA or DNA strand does not change the equilibrium constant (K(D)) for wild-type RT but the values of kinetic constants of association and dissociation differ significantly. For the p66(E478Q)/p51 enzyme, orientation effects are notable even altering the K(D) value. Binding of the p66(E478Q)/p51 to any RNA-DNA hybrids is slightly stronger compared with wild type. Data can be interpreted in terms of the mechanism of reverse transcription.
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PMID:HIV-1 reverse transcriptase interaction with model RNA-DNA duplexes. 1140 Dec 93

Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RTs of HIV-1 and HIV-2 are heterodimers of p66/p51 and p68/p54 subunits, respectively. The smaller subunit lacks the C-terminal segment of the larger subunit (which is the RNase H domain). The structure of the DNA polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains), linked to the RNase H domain via the connection subdomain. The RNase H activity of the Rod strain of HIV-2 RT is about tenfold lower than that of HIV-1 RT, while the DNA polymerase activity of these RTs is similar. A chimeric RT in which residues 227-427 (which constitute a small part of the palm and the entire thumb and connection subdomains) of the Rod strain of HIV-2 RT were replaced by the corresponding segment from HIV-1 RT, has an RNase H activity as high as HIV-1 RT (despite the fact that the RNase H domain is derived from HIV-2 RT). We analyzed the RNase H activity of wild-type HIV-2 RT from the D-194 strain and compared it with this activity of the RT from the Rod strain of HIV-2 and HIV-1 RT. The level of this activity of both HIV-2 RT strains was low; suggesting that low RNase H activity is a general property of HIV-2 isolates. The in vitro RNase H digestion pattern of the three wild-type RTs was indistinguishable, despite the difference in the level of RNase H activity. We constructed new chimeric HIV-1/HIV-2 RTs, in which protein segments and/or subunits were exchanged. The DNA polymerase activity of the parental HIV-1 and HIV-2 RTs was similar; as expected, the specific activity of the polymerases of all the hybrid RTs were also similar. However, the RNase H specific activity of the chimeric RTs was either high (like HIV-1 RT) or low (like HIV-2 RT). The origin of the thumb subdomain in the small subunit of the chimeric RTs (residues 244-322) determines the level of the RNase H activity. The strand-transfer activity of the chimeric RTs is also affected by the thumb subdomain of the small subunit; transfer was much more efficient if this subdomain was derived from HIV-1 RT. The data can be explained from the three-dimensional structure of HIV-1 RT. The thumb of the smaller subunit contacts the RNase H domain; it is through these contacts that the thumb affects the level of the RNase H activity of RT.
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PMID:The ribonuclease H activity of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2 is affected by the thumb subdomain of the small protein subunits. 1153 32

We present evidence that the integrases (INs) of HIV types 1 and 2 are inhibited in vitro by the reverse transcriptases (RTs) of HIV-1, HIV-2 and murine leukaemia virus. Both 3'-end processing and 3'-end joining (strand transfer) activities of IN were affected by the RTs. Full inhibitions were accomplished with most RT and IN combinations tested at around equimolar RT/IN ratios. The disintegration activity of IN was also inhibited by RTs. Neither DNA synthesis nor the ribonuclease H (RNase H) domain of RT were involved in IN inhibition, since specific DNA polymerase inhibitors did not affect the level of IN inhibition, and the p51 isoform of HIV-1 RT (which lacks the RNase H domain) is as effective in inhibiting IN as the heterodimeric p66/p51 isoform. On the other hand, the catalytic activities of HIV RTs were not affected by the INs, showing that RTs can inhibit IN activities, whereas INs do not inhibit RTs. We postulate that sequences and/or three-dimensional protein structures common to RTs interact with INs and inhibit their activities. We show evidence for this hypothesis and discuss the possible sites of IN involved in this interaction.
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PMID:Inhibition of the integrases of human immunodeficiency viruses type 1 and type 2 by reverse transcriptases. 1180 85

Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RT of human immunodeficiency virus type-1 (HIV-1) is composed of two subunits. The p51, which is the smaller subunit, shares with the larger p66 subunit the same amino-terminal part (which encompasses the DNA polymerase domain) and lacks the carboxyl-terminal segment of the p66 (which is the RNase H domain). The structure of the polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains) linked to the RNase H domain. Chemical modifications by thiol-specific reagents of cysteine 280, located in alpha helix I in the thumb subdomain of the polymerase domain, affect substantially only the RNase H activity. Also, the substitution of a serine for C280 did not alter any of the RT activities. Here we have systematically modified the C280 residue to either of the following residues: W, P, H, L, M, Y, Q, E or R. Only the first two mutations lead to a marked reduction in the RNase H activity, whereas none of the mutations affected the polymerase function to a significant extent. As expected, due to their impaired RNase H, the C280W and C280P mutants also had a very low DNA strand-transfer activity. It is also apparent from subunit-directed mutagenesis that each of the RT subunits contributes to the level of RNase H activity, yet the contribution of the p51 subunit to this activity is somewhat higher than that of the p66. Steady-state kinetic analyses have indicated that the RNase H activity was reduced mainly due to the sharp increase in the K(m) rather than changes in the k(cat) values. This suggests that the modifications of C280 lead to an impaired affinity of HIV-1 RT towards the RNA-DNA substrate.
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PMID:Mutagenesis of cysteine 280 of the reverse transcriptase of human immunodeficiency virus type-1: the effects on the ribonuclease H activity. 1261 5

Radiation target analysis is a powerful technique that can be used to determine both the structural and functional sizes of macromolecules. We have used this technique to probe the structure-function relationships of the recombinant forms of HIV-1 reverse transcriptase (RT). For the p66/p51 and p66/p66 dimeric forms of HIV-1 RT, both the structural and functional target sizes corresponded to that of the dimeric protein, indicating that a primary ionization in one subunit of the HIV-1 RT enzyme results in the concomitant polymer scission of both subunits. In contrast to p66/p51 and p66/p66 RT, the individually isolated p51 subunit of HIV-1 RT inactivated as a monomer. However, in the presence of a DNA template/primer substrate, the radiation inactivation analyses of p51 yielded a structural target size corresponding to that of a dimeric protein. This would indicate that the DNA substrate acted as a scaffold or template for p51 RT homodimer formation. In light of this observation, radiation inactivation studies can readily be applied to other DNA polymerase enzymes, such as the murine leukemia virus RT, for which the functional form of the enzyme has yet to be determined.
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PMID:Structure-activity relationships in HIV-1 reverse transcriptase revealed by radiation target analysis. 1293 Oct 6

A highly efficient cell-free translation system has been combined with suppressor tRNA technology to substitute nor-Tyr and 3-fluoro-Tyr in place of Tyr183 at the DNA polymerase active site of p66 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Supplementing the wild-type HIV-1 p51 RT subunit into this translation system permitted reconstitution of the biologically relevant p66/p51 heterodimer harboring Tyr analogs exclusively on the catalytically competent p66 subunit. Addition of an affinity tag at the p66 C-terminus allowed rapid, one-step purification of reconstituted and selectively mutated heterodimer HIV-1 RT via strep-Tactin-agarose affinity chromatography. The purified enzyme was demonstrated to be free of contaminating nucleases, allowing characterization of the DNA polymerase and ribonuclease H activities associated with HIV-1 RT. Preliminary characterization of HIV-1 RT(nor-Tyr) and HIV-1 RT(m-fluoro-Tyr) is presented. The success of this strategy will facilitate detailed molecular analysis of structurally and catalytically critical amino acids via their replacement with closely related, unnatural analogs.
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PMID:Site- and subunit-specific incorporation of unnatural amino acids into HIV-1 reverse transcriptase. 1547 80

To investigate how structural changes in the amino acid side chain affect nucleotide substrate selection in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a variety of non-natural tyrosine analogues were substituted for Tyr115 of p66 RT. RT variants containing meta-Tyr, nor-Tyr, aminomethyl-Phe, and 1- and 2-naphthyl-Tyr were produced in an Escherichia coli coupled transcription/translation system. Mutant p66 subunits were reconstituted with wild-type (WT) p51 RT and purified by affinity chromatography. Each modified enzyme retained DNA polymerase activity following this procedure. Aminomethyl-Phe115 RT incorporated dCTP more efficiently than the WT and was resistant to the chain terminator (-)-beta-2',3'-dideoxy-3'-thiacytidine triphosphate (3TCTP) when examined in a steady-state fidelity assay. However, 2-naphthyl-Tyr115 RT inefficiently incorporated dCTP at low concentrations and was kinetically slower with all dCTP analogues tested. Models of RT containing these side chains suggest that the aminomethyl-Phe115 substitution provides new hydrogen bonds through the minor groove to the incoming dNTP and the template residue of the terminal base pair. These hydrogen bonds likely contribute to the increased efficiency of dCTP incorporation. In contrast, models of HIV-1 RT containing 2-naphthyl-Tyr115 reveal significant steric clashes with Pro157 of the p66 palm subdomain, necessitating rearrangement of the active site.
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PMID:Investigating the "steric gate" of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by targeted insertion of unnatural amino acids. 1727 99

Several rare and novel NNRTI [non-nucleoside reverse transcriptase (RT) inhibitor] resistance mutations were recently detected at codons 132 and 135 in RTs from clinical isolates using the yeast-based chimaeric TyHRT (Ty1/HIV-1 RT) phenotypic assay. Ile132 and Ile135 form part of the beta7-beta8 loop of HIV-1 RT (residues 132-140). To elucidate the contribution of these residues in RT structure-function and drug resistance, we constructed twelve recombinant enzymes harbouring mutations at codons 132 and 135-140. Several of the mutant enzymes exhibited reduced DNA polymerase activities. Using the yeast two-hybrid assay for HIV-1 RT dimerization we show that in some instances this decrease in enzyme activity could be attributed to the mutations, in the context of the 51 kDa subunit of HIV-1 RT, disrupting the subunit-subunit interactions of the enzyme. Drug resistance analyses using purified RT, the TyHRT assay and antiviral assays demonstrated that the I132M mutation conferred high-level resistance (>10-fold) to nevirapine and delavirdine and low-level resistance (approximately 2-3-fold) to efavirenz. The I135A and I135M mutations also conferred low level NNRTI resistance (approximately 2-fold). Subunit selective mutagenesis studies again demonstrated that resistance was conferred via the p51 subunit of HIV-1 RT. Taken together, our results highlight a specific role of residues 132 and 135 in NNRTI resistance and a general role for residues in the beta7-beta8 loop in the stability of HIV-1 RT.
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PMID:Characterization of novel non-nucleoside reverse transcriptase (RT) inhibitor resistance mutations at residues 132 and 135 in the 51 kDa subunit of HIV-1 RT. 1728 55

Previous studies have demonstrated that nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) act as chemical enhancers of human immunodeficiency virus type 1 (HIV-1) RT dimerization. In the current study, we sought to define the role of key residues (101, 103, 108, 181, 188, 190, 225 and 318) in the NNRTI-binding pocket on HIV-1 RT heterodimer stability. Thirteen mutant RTs were constructed and evaluated for p66/p51 RT heterodimer formation using the well-established yeast two-hybrid assay. We found that the mutations K101A, P225H, Y318F and Y318W decreased RT heterodimer stability whereas K103N, V108I, V108W, Y181C, Y188L, G190A, G190E, G190W and P225W increased RT heterodimer stability. While these results demonstrate that residues that comprise the NNRTI-binding pocket contribute to the stability of p66/p51 HIV-1 RT, they did not suggest any obvious correlation between RT dimer stability and the extent of NNRTI resistance. Remarkably, mutations at residue G190 (A, E, W) in the p66 RT subunit were found to dramatically increase heterodimer stability. Notably, the G190W mutation increased RT dimer stability almost to the same extent as did 5 microM efavirenz. In light of these findings, we characterized the in vitro activity of recombinant RT expressing mutations at G190 in the p66 subunit only and compared them with a wild-type enzyme complexed with efavirenz. We found that while mutations at G190 had a significant effect on both the DNA polymerase and ribonuclease H activity of the enzyme, their phenotypic effects did not mirror those induced by efavirenz-binding to RT.
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PMID:Impact of residues in the nonnucleoside reverse transcriptase inhibitor binding pocket on HIV-1 reverse transcriptase heterodimer stability. 1833 60

High-throughput screening of National Cancer Institute libraries of synthetic and natural compounds identified the vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4 H-cyclohepta[ b]thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT). A Yonetani-Theorell analysis demonstrated that NSC727447, and the active-site hydroxytropolone RNase H inhibitor beta-thujaplicinol were mutually exclusive in their interaction with the RNase H domain. Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the thumb subdomain of p51 were affected by ligand binding. Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb. Site-directed mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or non-natural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity. NSC727447 thus represents a novel class of RNase H antagonists with a mechanism of action differing from active site, divalent metal-chelating inhibitors that have been reported.
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PMID:Vinylogous ureas as a novel class of inhibitors of reverse transcriptase-associated ribonuclease H activity. 1883 89


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