Mechanism of phosphonoacetate inhibition of herpesvirus-induced DNA polymerase

Leinbach, Susan S.; Reno, John M.; Lee, Lucy F.; Isbell, A. F.; Boezi, John A.

doi:10.1021/bi00647a029

Cited by 209 publications

(84 citation statements)

References 21 publications

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“…PAA appears to act as a pyrophosphate analog in the polymerase active center of sensitive DNA polytial to be useful, but the currently available inhibitors merases to severely reduce the polymerization reaction are not specific. Aphidicolin, for example, inhibits all (Leinbach et al 1976). Thus, since yeast like other euthree replicative DNA polymerases, DNA pols ␣, ␦, and karyotes is relatively resistant to PAA, PAA sensitivity is ε (Burgers and Bauer 1988).…”

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confidence: 99%

Sensitivity to Phosphonoacetic Acid

Li¹,

Murphy²,

Kanevets

et al. 2005

Genetics

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A mutant allele (pol3-L612M) of the DNA polymerase ␦ 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 ␦ requires Rad27 flap endonuclease activity since the pol3-L612M strain is not viable in the absence of RAD27 function. The L612M-DNA pol ␦ 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. E UKARYOTIC DNA polymerase ␦ (DNA pol ␦) is reacid (foscarnet) are effective antiviral drugs that inhibit replication by herpes and vaccinia DNA polymerases quired for chromosome replication, recombination, and repair, but there are several other DNA polymerases (Mao et al. 1975;Ö berg 1989;Taddie and Traktman 1991) and the HIV reverse transcriptase (Larder et al. in the cell, which makes it difficult to study DNA pol ␦ specifically. DNA polymerase inhibitors have the poten-1987). PAA appears to act as a pyrophosphate analog in the polymerase active center of sensitive DNA polytial to be useful, but the currently available inhibitors merases to severely reduce the polymerization reaction are not specific. Aphidicolin, for example, inhibits all (Leinbach et al. 1976). Thus, since yeast like other euthree replicative DNA polymerases, DNA pols ␣, ␦, and karyotes is relatively resistant to PAA, PAA sensitivity is ε (Burgers and Bauer 1988). Mutations that confer expected to increase substantially if the wild-type DNA temperature sensitivity (ts) provide a way to block replipol ␦ is converted to a PAA-sensitive mutant. cation by a selected DNA polymerase, but ts DNA pol ␦ To construct a yeast DNA pol ␦ mutant with PAA sensimutants lose viability rapidly after exposure to the restrictivity, we used mutational studies of the bacteriophage T4 tive temperature (Weinert and Hartwell 1993), which DNA polymerase as a guide (Reha-Krantz 1995). The prevents studies of recovery mechanisms. We report a bacteriophage T4 DNA polymerase, like eukaryotic DNA new method for inhibiting DNA pol ␦ selectively: we pols ␣, ␦, and ε, is relatively resistant to PAA; however, constructed a mutant DNA pol ␦ in Saccaromyces cerevisiae several mutant T4 DNA polymerases were identified that is inhibited by the antiviral drug phosphonoacetic with markedly increased sensitivity (Reha-Krantz et al. acid (PAA).1993; Reha-Krantz and Nonay 1994). T4 DNA poly-PAA was chosen as a new DNA pol ␦ inhibitor primarmerase and eukaryotic DNA pol ␦ are members of a ily for two reasons. First, PAA does not need to be prol...

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confidence: 99%

Sensitivity to Phosphonoacetic Acid

Li¹,

Murphy²,

Kanevets

et al. 2005

Genetics

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“…Results from biochemical (e.g. Leinbach et al, 1976;Reno et al, 1978) and genetic (Honess & Watson, 1977;Chartrand et al, 1979Chartrand et al, , 1980 investigations are consistent with inhibition of the activity of the virus-coded DNA polymerase as the primary site of action of PAA on HSV replication. Phosphonoacetic acid-resistant (U) variants of HSV are readily selected during virus growth in the presence of the drug and pr determinants have been linked by both conventional genetic (Honess & Watson, 1977; and physical mapping techniques to the DNA polymerase locus of HSV.…”

Section: Introductionmentioning

confidence: 69%

Complementation between Phosphonoacetic Acid-resistant and -sensitive Variants of Herpes Simplex Viruses: Evidence for an Oligomeric Protein with Restricted Intracellular Diffusion as the Determinant of Resistance and Sensitivity

Honess¹

1981

Journal of General Virology

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SUMMARYComplementation between phosphonoacetic acid (PAA)-resistant (pr) and -sensitive (ps) variants of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) was studied to provide information on the function of the virus-coded DNA polymerase. Complementation within and between serotypes was demonstrated, with the growth of the ps partner in mixed infections becoming relatively more resistant and the pr partner relatively more sensitive to PAA than in the corresponding single infections. However, the relative contribution of the ps partner to the mixed infection had a disproportionately large effect on the resultant sensitivity of the mixed infection which was incompatible with nonqnteractive (e.g. monomeric) polymerase molecules as determinants of PAA sensitivity and resistance. Although a number of solutions gave equally good fits to the available data, the simplest was obtained by assuming that the functional DNA polymerase was a trimer and that only the (pr)3 homotrimer was active in the presence of the drug. In addition, yields from mixed infections in the presence of PAA were enriched for the resistant partner relative to yields in the absence of the drug. These latter results suggested that the intracellular distribution of resistant DNA polymerase oligomers was non-random with respect to resistant and sensitive template genomes and that these resistant polymerase molecules were more likely to encounter and replicate resistant than sensitive genomes. Such an explanation seems to require vectorial nuclear-cytoplasm-nucleus translocation and restricted diffusion of transcript and gene products determining resistance.

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“…These studies lead to the conclusion that PFA does not compete with the incoming nucleotide. On the other hand, it has been consistently found that PFA competitively inhibited the ATP-PP i exchange reaction (26,29), and PFA has been used as a pyrophosphate analog for product inhibition studies in a variety of DNA polymerases (29,30). The interaction between foscarnet and pyrophosphate is not completely straightforward, however, because high levels of resistance to foscarnet are usually not associated with high levels of resistance to PP i (14,26,28).…”

Section: Discussionmentioning

confidence: 99%

Selective Excision of Chain-terminating Nucleotides by HIV-1 Reverse Transcriptase with Phosphonoformate as Substrate

Cruchaga

Ansó

Rouzaut

et al. 2006

Journal of Biological Chemistry

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A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP i ) analogs on PP i -and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP i -and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT⅐template/primer complex was hindered by the presence of a chain terminator at the 3-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZTtriphosphate in the presence of PP i or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.

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Mechanism of phosphonoacetate inhibition of herpesvirus-induced DNA polymerase

Cited by 209 publications

References 21 publications

Sensitivity to Phosphonoacetic Acid

Sensitivity to Phosphonoacetic Acid

Complementation between Phosphonoacetic Acid-resistant and -sensitive Variants of Herpes Simplex Viruses: Evidence for an Oligomeric Protein with Restricted Intracellular Diffusion as the Determinant of Resistance and Sensitivity

Selective Excision of Chain-terminating Nucleotides by HIV-1 Reverse Transcriptase with Phosphonoformate as Substrate

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