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)

Structures of the 31-kilodalton catalytic domain of rat DNA polymerase beta (pol beta) and the whole 39-kilodalton enzyme were determined at 2.3 and 3.6 angstrom resolution, respectively. The 31-kilodalton domain is composed of fingers, palm, and thumb subdomains arranged to form a DNA binding channel reminiscent of the polymerase domains of the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, and bacteriophage T7 RNA polymerase. The amino-terminal 8-kilodalton domain is attached to the fingers subdomain by a flexible hinge. The two invariant aspartates found in all polymerase sequences and implicated in catalytic activity have the same geometric arrangement within structurally similar but topologically distinct palms, indicating that the polymerases have maintained, or possibly re-evolved, a common nucleotidyl transfer mechanism. The location of Mn2+ and deoxyadenosine triphosphate in pol beta confirms the role of the invariant aspartates in metal ion and deoxynucleoside triphosphate binding.
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PMID:Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. 751 81

Replication complexes that contained either murine leukemia virus reverse transcriptase (MLV RT) or a variant reverse transcriptase without a ribonuclease (RNase) H domain (delta RH MLV RT) were visualized by enzymatic footprinting. Wild-type MLV RT protected template nucleotides +6 to -27, and primer nucleotides -1 to -26 of primers that had first been extended by one or four nucleotides. Although it catalyzed DNA synthesis, delta RH MLV RT stably bound template-primer only under conditions of reduced ionic strength and protected the duplex portion only as far as position -15. Despite altered hydrolysis profiles, both enzymes covered primarily the template-primer duplex, contradicting recent predictions based on the structure of rat DNA polymerase beta.
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PMID:Footprint analysis of replicating murine leukemia virus reverse transcriptase. 752 42

(-)-beta-L-2',3'-Dideoxycytidine (L-ddC) and (-)-beta-L-2',3'-dideoxy-5-fluorocytidine (L-FddC) have been reported to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in vitro. In the present study, the 5'-triphosphates of L-ddC (L-ddCTP) and L-FddC (L-FddCTP) were demonstrated to competitively inhibit HIV-1 reverse transcriptase (RT), with inhibition constants (KiS) of 2 and 1.6 microM, respectively, when a poly(rI).oligo(dC)10-15 template primer was used; in comparison Ki values for beta-D-2',3'-dideoxycytidine 5'-triphosphate (D-ddCTP) and beta-D-2',3'-dideoxy-5-fluorocytidine 5'-triphosphate (D-FddCTP) were 1.1 and 1.4 microM, respectively. Use of the mutant RT at position 184 (substitution of methionine to valine [M184V]), which is associated with resistance to beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), resulted in significant increases (50- to 60-fold) in Ki values for L-ddCTP and L-FddCTP, whereas the elevation in Ki values for D-ddCTP and D-FddCTP was moderate (2-fold). L-ddCTP and L-FddCTP did not inhibit human DNA polymerases alpha and beta up to 100 microM. In contrast, D-ddCTP and D-FddCTP inhibited human DNA polymerase beta, with Ki values of 0.5 and 2.5 microM, respectively. By using sequencing analysis, L-ddCTP and L-FddCTP exhibited DNA chain-terminating activities toward the parental HIV-1 RT, whereas they were not a substrate for the mutant M184V HIV-1 RT.L-ddC and L-FddC did not inhibit the mitochondrial DNA content of human cells up to a concentration of 10 microM, whereas D-ddC and D-FddC decreased the mitochondrial DNA content by 90% at concentrations of 1 and 10 microM, respectively. All of these results suggest that further development of L-ddC, and L-FddC in particular, is warranted as a possible anti-HIV candidate.
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PMID:Inhibition of human immunodeficiency virus type 1 reverse transcriptase by the 5'-triphosphate beta enantiomers of cytidine analogs. 753 Sep 32

DNA and RNA polymerases are enzymes that are primarily responsible for copying genetic material in all living systems. The four polymerases whose structures have been determined by X-ray crystallographic methods have significant similarities at the polymerase active site that are indicative of common requirements for polynucleotide synthesis. Structural studies of complexes of the Klenow fragment of Escherichia coli DNA polymerase I, HIV type 1 reverse transcriptase, and rat DNA polymerase beta with DNA are leading to generalized models for catalysis.
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PMID:Structures of DNA and RNA polymerases and their interactions with nucleic acid substrates. 753 8

L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.
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PMID:Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate. 754 86

5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-beta-D- lyxofuranosyl)thymine, 1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl)thymine and 2',3'-lyxoanhydrothymidine have been shown to be termination substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) reverse transcriptases as well as DNA polymerase I from E. coli and DNA polymerase beta from rat liver. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase epsilon from human placenta. Km values of ltTTP, rtTTP and laTTP incorporation into the DNA chain during catalysis by AMV reverse transcriptase agree closely with each other being 1.5-2.5 times higher than Km value for dTTP. Furthermore, Vmax values for modified substrates are only 2-3 times lower than Vmax for dTTP. The evidence favours the hypothesis of high affinity of modified nucleotides with a flattened furanosyl ring for DNA polymerase active sites.
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PMID:Modified nucleoside 5'-triphosphates containing 2',3'-fused three-membered rings as substrates for different DNA polymerases. 768 65

Eukaryotic DNA polymerase beta and the reverse transcriptases are the most inaccurate of the known DNA polymerases. We report here mutagenic replication in vitro past intrastrand N(7)G-N(7)G chelates of the cis-diamminedichloroplatinum(II), the major DNA adduct of the antitumor agent cisplatin by calf thymus DNA polymerase beta and human immunodeficiency virus type I reverse transcriptase (42% and 26% mutations, respectively). The most frequent modifications generated by both enzymes were one-base frameshift deletions. Only one mutational hot spot opposite the platinated guanines was observed with human immunodeficiency virus type I reverse transcriptase, while two hot spots were generated by DNA polymerase beta, one at the base situated 5' to the lesion and the other situated 4-6 nucleotides 5' to the adduct. An unusual mutagenic event, tandem replication of a 12-base pair sequence, was observed with DNA polymerase beta. The mutational spectra of the two DNA polymerases suggest that template slippage occurred with higher frequency in the presence of the more distributive DNA polymerase beta.
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PMID:In vitro bypass replication of the cisplatin-d(GpG) lesion by calf thymus DNA polymerase beta and human immunodeficiency virus type I reverse transcriptase is highly mutagenic. 866 82

The dNTP binding pocket of human immunodeficiency virus type 1 reverse transcriptase (RT) and DNA polymerase beta (beta-pol) were labeled using a photoreactive analog of dCTP, exo-N-[beta-(p-azidotetrafluorobenzamido)-ethyl]-deoxycytidine-5'- triphosphate (FABdCTP). Two approaches of photolabeling were utilized. In one approach, photoreactive FABdCTP and radiolabeled primer-template were UV-irradiated in the presence of each enzyme and resulted in polymerase radiolabeling. In an alternate approach, FABdCTP was first UV-cross-linked to enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked dCTP analog was incorporated onto the 3'-end of the radiolabeled primer. The results showed strong labeling of the p66 subunit of RT, with only minor labeling of p51. No difference in the intensity of cross-linking was observed with either approach. FABdCTP cross-linking was increased in the presence of a dideoxyterminated primer-template with RT, but not with beta-pol, suggesting a significant influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of beta-pol residues observed to interact with the incoming dNTP in the crystal structure of the ternary complex resulted in labeling consistent with kinetic characterization of these mutants and indicated specific labeling of the dNTP binding pocket.
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PMID:dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase beta as revealed by affinity labeling with a photoreactive dNTP analog. 870 91

The recently developed ultrasensitive reverse transcriptase (RT) test involving a PCR step can detect minute amounts of RT in single retroviral particles and is 10(6) times more sensitive than conventional RT assays. We have found that different DNA-dependent DNA polymerases like DNA Polymerase I from Escherichia coli and eukaryotic enzymes like DNA polymerase alpha and gamma exhibit RT-like activities in this assay, whereas DNA polymerase beta and Klenow enzyme show only minor activities. To discriminate false-positive signals caused by DNA-dependent DNA polymerases in the RT-PCR assay, we have included increasing amounts of activated DNA in the RT reactions. This modification of the assay leads to complete inhibition of aberrant but not authentic RT activities. This improvement specifies the RT-PCR assay as the most sensitive tool for the detection of even very rare replication-competent retroviruses and of related enzymatic activities indigenous, for example, for products of endogenous retrovirus-like RT genes in cell extracts.
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PMID:Specific suppression of false-positive signals in the product-enhanced reverse transcriptase assay. 882 50

Some natural and glycon-modified dNTPs with beta,gamma-pyrophosphate substitution at the triphosphate residue were synthesized and studied to evaluate the effect of these modifications on substrate properties of dNTPs in DNA synthesis catalyzed by human placental DNA polymerases alpha and beta, avian myeloblastosis virus reverse transcriptase, and calf thymus terminal deoxynucleotidyl transferase. Reverse transcriptase proved to be the enzyme least specific to such modifications; the substrate activity of beta,gamma-methylenediphosphonate substituted dTTP and 3'-azido-3'-deoxy-dTTP decreased in the following order: CF2 = CHF > CBr2 > CFMe >> CH2. This order is individual for each DNA polymerase. It is interesting to mention that beta,gamma-CBr2 substituted dTTP is neither a substrate nor an inhibitor of DNA polymerase beta. This specificity distinguishes DNA polymerase beta from other DNA polymerases studied.
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PMID:Effect of triphosphate modifications in 2'-deoxynucleoside 5'-triphosphates on their specificity towards various DNA polymerases. 923 75


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