Sensitivity to Phosphonoacetic Acid

Abstract

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...

Mutator Phenotypes Caused by Substitution at a Conserved Motif A Residue in Eukaryotic DNA Polymerase δ

“…The largest elevations were obtained for L612K (13-fold), L612G (17-fold), and L612N (37-fold), the increase for L612N being similar to that seen for the pol3-01 mutant lacking exonucleolytic proofreading (29-fold) (29,32,43). The increase for L612M (7-fold) is in accord with the 3.5-fold elevation recently reported (44). Interestingly, the increase in mutation rate was not strictly correlated with other phenotypic deficiencies.…”

“…Based on the relative replication proficiency and the predominance of base substitutions in the mutation spectrum (86%; Table 2), we hypothesized that the L612M mutator phenotype may reflect mainly errors made by the polymerase, rather than secondary mutagenic processes induced by defective replication. The recent work of Li et al (44), which was published as we completed this manuscript, sheds light on the function of the L612M polymerase. These authors also observed a mutator phenotype; the rate of forward mutation at CAN1 was elevated 3.5-fold, consistent with our results.…”

Phosphonoformic Acid Inhibits Viral Replication by Trapping the Closed Form of the DNA Polymerase

“…Many questions concerning the exact binding site of PFA have remained elusive, however, in part because poor expression levels of recombinant UL54 have precluded prior crystallographic investigation. Moreover, it is still unclear why PFA preferentially inhibits herpesviridae DNA polymerases, whereas other family B polymerases, such as RB69 DNA polymerase (gp43) or the replicative eukaryotic DNA polymerases ␦ or ⑀, are left unaffected by the drug (11).…”

Contrasting Effects of W781V and W780V Mutations in Helix N of Herpes Simplex Virus 1 and Human Cytomegalovirus DNA Polymerases on Antiviral Drug Susceptibility

“…Contrary to the findings for UL30 and UL54 DNA polymerases, the gp43 enzyme is relatively resistant to foscarnet (10). Like all ␣-like DNA polymerases, the RB69 enzyme in binary complex with DNA adopts the open conformation for the fingers do- Replicative capacity of wild-type virus and two independent recombinant HCMV strains harboring mutation W780V in HFFs determined by the GLuc reporter-based assay.…”

“…Despite a high degree of similarity with the UL30 and UL54 DNA polymerases, the gp43 enzyme is relatively resistant to the broad-spectrum antiviral agent foscarnet (10). It was suggested that the binding site for foscarnet on the UL54 DNA polymerase is located in close proximity to the ␥ phosphate of the bound nucleotide (11).…”