The Experts below are selected from a list of 105 Experts worldwide ranked by ideXlab platform
João V. Costa - One of the best experts on this subject based on the ideXlab platform.
-
Functional and molecular characterization of African swine fever virus mutants resistant to Phosphonoacetic Acid.
Virology, 1995Co-Authors: Maria Isabel Marques, João V. CostaAbstract:African swine fever virus (ASFV) growth and plaque formation were inhibited by Phosphonoacetic Acid (PAA) concentrations of 200 micrograms/ml or more. One spontaneous mutant and two mutants isolated from mutagenized virus were resistant to PAA inhibition and showed practically normal viral DNA synthesis in the presence of PAA. DNA polymerase activity present in the cytoplasmic fraction from cells infected with the mutants required 10-fold higher concentrations of PAA for inhibition compared to equivalent inhibition of the wild-type enzyme. Like wild-type virus, the PAA-resistant mutants were resistant to inhibition by aphidicolin. Marker rescue analysis with mutant DNA fragments covering different regions of the ASFV DNA polymerase gene mapped the mutations within a fragment which was cloned and sequenced. A single nucleotide and amino Acid change was assigned to each mutant. Two of the PAA-resistant mutations lie within the highly conserved region II common to alpha-like DNA polymerases, which has been implicated in pyrophosphate binding and probably also in dNTP binding. The other mutation was localized to within a region of moderate homology among viral DNA polymerases close to one of the motifs allegedly considered as constituting the 3'-5' exonuclease active site.
-
African swine fever virus-induced DNA polymerase is resistant to aphidicolin.
Virology, 1992Co-Authors: Maria Isabel Marques, João V. CostaAbstract:Abstract African swine fever virus (ASFV) induces the synthesis of a virus-specific DNA polymerase, which is inhibited by Phosphonoacetic Acid and cytosine arabinoside. In contrast to all other a-like DNA polymerases of DNA viruses, ASFV-specific DNA polymerase is resistant to aphidicolin. Concentrations of the drug as high as 160 μM had no effect on virus production or plaquing efficiency. The resistance of ASFV DNA polymerase to aphidicolin was confirmed by analyzing the effect of the drug on viral DNA synthesis. A moderate inhibition of viral DNA synthesis was observed when aphidicolin was added immediately after virus adsorption but normal synthesis occurred, with a peak at 10 hr p.i., when the drug was added at 2 or 4 hr p.i. This suggests that a very early phase of ASFV DNA replication is sensitive to aphidicolin and is probably catalyzed by a different enzyme. An in vitro assay of DNA polymerase activity was used to assay the sensitivity of the virus-specific DNA polymerase to inhibitors. In correspondence to the results observed in vivo , Phosphonoacetic Acid strongly inhibited the enzyme activity, whereas aphidicolin had no effect. Resistance to aphidicolin was independent of the concentration of dCTP used in the assay. Three independent ASFV mutants resistant to Phosphonoacetic Acid showed the same resistance to aphidicolin as wild type virus.
Maria Isabel Marques - One of the best experts on this subject based on the ideXlab platform.
-
Functional and molecular characterization of African swine fever virus mutants resistant to Phosphonoacetic Acid.
Virology, 1995Co-Authors: Maria Isabel Marques, João V. CostaAbstract:African swine fever virus (ASFV) growth and plaque formation were inhibited by Phosphonoacetic Acid (PAA) concentrations of 200 micrograms/ml or more. One spontaneous mutant and two mutants isolated from mutagenized virus were resistant to PAA inhibition and showed practically normal viral DNA synthesis in the presence of PAA. DNA polymerase activity present in the cytoplasmic fraction from cells infected with the mutants required 10-fold higher concentrations of PAA for inhibition compared to equivalent inhibition of the wild-type enzyme. Like wild-type virus, the PAA-resistant mutants were resistant to inhibition by aphidicolin. Marker rescue analysis with mutant DNA fragments covering different regions of the ASFV DNA polymerase gene mapped the mutations within a fragment which was cloned and sequenced. A single nucleotide and amino Acid change was assigned to each mutant. Two of the PAA-resistant mutations lie within the highly conserved region II common to alpha-like DNA polymerases, which has been implicated in pyrophosphate binding and probably also in dNTP binding. The other mutation was localized to within a region of moderate homology among viral DNA polymerases close to one of the motifs allegedly considered as constituting the 3'-5' exonuclease active site.
-
African swine fever virus-induced DNA polymerase is resistant to aphidicolin.
Virology, 1992Co-Authors: Maria Isabel Marques, João V. CostaAbstract:Abstract African swine fever virus (ASFV) induces the synthesis of a virus-specific DNA polymerase, which is inhibited by Phosphonoacetic Acid and cytosine arabinoside. In contrast to all other a-like DNA polymerases of DNA viruses, ASFV-specific DNA polymerase is resistant to aphidicolin. Concentrations of the drug as high as 160 μM had no effect on virus production or plaquing efficiency. The resistance of ASFV DNA polymerase to aphidicolin was confirmed by analyzing the effect of the drug on viral DNA synthesis. A moderate inhibition of viral DNA synthesis was observed when aphidicolin was added immediately after virus adsorption but normal synthesis occurred, with a peak at 10 hr p.i., when the drug was added at 2 or 4 hr p.i. This suggests that a very early phase of ASFV DNA replication is sensitive to aphidicolin and is probably catalyzed by a different enzyme. An in vitro assay of DNA polymerase activity was used to assay the sensitivity of the virus-specific DNA polymerase to inhibitors. In correspondence to the results observed in vivo , Phosphonoacetic Acid strongly inhibited the enzyme activity, whereas aphidicolin had no effect. Resistance to aphidicolin was independent of the concentration of dCTP used in the assay. Three independent ASFV mutants resistant to Phosphonoacetic Acid showed the same resistance to aphidicolin as wild type virus.
Linda J. Reha-krantz - One of the best experts on this subject based on the ideXlab platform.
-
Sensitivity to Phosphonoacetic Acid: A New Phenotype to Probe DNA Polymerase δ in Saccharomyces cerevisiae
Genetics, 2005Co-Authors: Lei Li, Kelly Murphy, Uliana Kanevets, Linda J. Reha-krantzAbstract:A mutant allele (pol3-L612M) of the DNA polymerase δ gene in Saccharomyces cerevisiae that confers sensitivity to the antiviral drug Phosphonoacetic Acid (PAA) was constructed. We report that PAA-sensitivity tagging DNA polymerases is a useful method for selectively and reversibly inhibiting one type of DNA polymerase. Our initial studies reveal that replication by the L612M-DNA pol δ requires Rad27 flap endonuclease activity since the pol3-L612M strain is not viable in the absence of RAD27 function. The L612M-DNA pol δ also strongly depends on mismatch repair (MMR). Reduced viability is observed in the absence of any of the core MMR proteins—Msh2, Mlh1, or Pms1—and severe sensitivity to PAA is observed in the absence of the core proteins Msh6 or Exo1, but not Msh3. We propose that pol3-L612M cells need the Rad27 flap endonuclease and MMR complexes composed of Msh2/Msh6, Mlh1/Pms1, and Exo1 for correct processing of Okazaki fragments.
-
Selection of bacteriophage T4 antimutator DNA polymerases: a link between proofreading and sensitivity to Phosphonoacetic Acid
Mutation research, 1996Co-Authors: Linda J. Reha-krantz, Cindy WongAbstract:During DNA replication, DNA polymerases alternate between DNA synthesis and proofreading the newly synthesized DNA. In order to understand the molecular details of how DNA polymerases determine the balance between polymerase and proofreading activities, it would be useful to have mutants which switch between the two activities either more or less frequently. Antimutator DNA polymerases switch more frequently and thus have more opportunity for proofreading. We have observed that mutant DNA polymerases which proofread less frequently have a mutator phenotype and are inhibited by the pyrophosphate analogue Phosphonoacetic Acid. Sensitivity to Phosphonoacetic Acid can be used to isolate second-site suppressor mutations. These suppressor mutations encode amino Acid substitutions which produce antimutator DNA polymerases.
-
Bacteriophage T4 DNA polymerase mutations that confer sensitivity to the PPi analog Phosphonoacetic Acid.
Journal of virology, 1993Co-Authors: Linda J. Reha-krantz, R L Nonay, S StockiAbstract:Mutations that conferred sensitivity to the pyrophosphate analog Phosphonoacetic Acid in bacteriophage T4 DNA polymerase were identified. The mutations were loosely clustered in four regions of the gene. As found for herpes simplex virus DNA polymerase, T4 mutations that altered sensitivity to Phosphonoacetic Acid also altered sensitivity to nucleotide analogs. Some of the T4 DNA polymerase mutations also altered the ability of the enzyme to translocate from one template position to the next and affected DNA replication fidelity. Kornberg (A. Kornberg, Science 163:1410-1418, 1969) envisioned a DNA polymerase active center which accommodates primer terminus and template DNAs and the incoming nucleotide. Some mutations identified on the basis of sensitivity to Phosphonoacetic Acid may be part of such an active center because single amino Acid substitutions simultaneously alter several DNA polymerase functions.
Likui Zhang - One of the best experts on this subject based on the ideXlab platform.
-
New Insights into DNA Polymerase Function Revealed by Phosphonoacetic Acid-Sensitive T4 DNA Polymerases
Chemical research in toxicology, 2017Co-Authors: Likui ZhangAbstract:The bacteriophage T4 DNA polymerase (pol) and the closely related RB69 DNA pol have been developed into model enzymes to study family B DNA pols. While all family B DNA pols have similar structures and share conserved protein motifs, the molecular mechanism underlying natural drug resistance of nonherpes family B DNA pols and drug sensitivity of herpes DNA pols remains unknown. In the present study, we constructed T4 phages containing G466S, Y460F, G466S/Y460F, P469S, and V475W mutations in DNA pol. These amino Acid substitutions replace the residues in drug-resistant T4 DNA pol with residues found in drug-sensitive herpes family DNA pols. We investigated whether the T4 phages expressing the engineered mutant DNA pols were sensitive to the antiviral drug Phosphonoacetic Acid (PAA) and characterized the in vivo replication fidelity of the phage DNA pols. We found that G466S substitution marginally increased PAA sensitivity, whereas Y460F substitution conferred resistance. The phage expressing a double muta...
Yoshimitsu Nagao - One of the best experts on this subject based on the ideXlab platform.
-
e selective horner wadsworth emmons reaction of aryl alkyl ketones with bis 2 2 2 trifluoroethyl Phosphonoacetic Acid
Tetrahedron Letters, 2003Co-Authors: Shigeki Sano, Yuka Takemoto, Yoshimitsu NagaoAbstract:Abstract The stereoselective Horner–Wadsworth–Emmons reaction of aryl alkyl ketones with bis(2,2,2-trifluoroethyl)Phosphonoacetic Acid utilizing lithium hexamethyldisilazide in DMF afforded ( E )-α,β-unsaturated carboxylic Acids as the major products.
-
(E)-Selective Horner–Wadsworth–Emmons reaction of aldehydes with bis-(2,2,2-trifluoroethyl)Phosphonoacetic Acid
Arkivoc, 2003Co-Authors: Shigeki Sano, Yuka Takemoto, Yoshimitsu NagaoAbstract:The stereoselective Horner–Wadsworth–Emmons reaction of aldehydes with bis-(2,2,2trifluoroethyl)Phosphonoacetic Acid utilizing i-PrMgBr afforded (E)-α,β-unsaturated carboxylic Acids as the major products. bis-(2,2,2-Trifluoroethyl)Phosphonoacetic Acid was prepared by enzymatic hydrolysis of the corresponding methyl ester with porcine liver esterase.
-
e selective horner wadsworth emmons reaction of aldehydes with bis 2 2 2 trifluoroethyl Phosphonoacetic Acid
Arkivoc, 2003Co-Authors: Shigeki Sano, Yuka Takemoto, Yoshimitsu NagaoAbstract:The stereoselective Horner–Wadsworth–Emmons reaction of aldehydes with bis-(2,2,2trifluoroethyl)Phosphonoacetic Acid utilizing i-PrMgBr afforded (E)-α,β-unsaturated carboxylic Acids as the major products. bis-(2,2,2-Trifluoroethyl)Phosphonoacetic Acid was prepared by enzymatic hydrolysis of the corresponding methyl ester with porcine liver esterase.
-
(E)-Selective Horner–Wadsworth–Emmons reaction of aryl alkyl ketones with bis(2,2,2-trifluoroethyl)Phosphonoacetic Acid
Tetrahedron Letters, 2003Co-Authors: Shigeki Sano, Yuka Takemoto, Yoshimitsu NagaoAbstract:Abstract The stereoselective Horner–Wadsworth–Emmons reaction of aryl alkyl ketones with bis(2,2,2-trifluoroethyl)Phosphonoacetic Acid utilizing lithium hexamethyldisilazide in DMF afforded ( E )-α,β-unsaturated carboxylic Acids as the major products.