Inhibition of hepatitis B Dane particle DNA polymerase activity by pyrophosphate analogs

A DNA polymerase is associated with the core of the so-called Dane particles. The probability that this is the hepatitis B viral DNA polymerase offers the possibility of preventing hepatitis B multiplication by selective inhibition of this enzyme. We have previously reported that trisodium phosphonoformate (PFA) inhibits Dane particle DNA polymerase. Fifteen compounds with structural similarity to PFA and pyrophosphate have now been tested for inhibition of hepatitis B virus DNA polymerase in an attempt to define the structural requirement for the inhibition. Active structures have two acid groups at close proximity of which at least one is a phosphono group. Phosphonoformate and hypophosphare were the two most active inhibitors. The Ki value for PFA was 7.2 microM when dTTP was used as variable substrate, and the mechanism of inhibition was non-competitive. Phosphonoformate caused rapid shut-off of the polymerase reaction, indicating that it might inhibit elongation. The efficient inhibition of hepatitis B virus DNA polymerase by PFA and its low toxicity suggest that it could be used to inhibit hepatitis B virus multiplication in vivo.     

Termination of DNA synthesis by 9-beta-D-arabinofuranosyl-2-fluoroadenine. A mechanism for cytotoxicity.

The action of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on DNA synthesis was evaluated both in whole cells and in vitro. 9-beta-D-Arabinofuranosyl-2-fluoroadenine was converted to its 5'-triphosphate 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) in cells and then incorporated into DNA in a self-limiting manner. More than 94% of the analogue was incorporated into DNA at the 3' termini, indicating a chain termination action. In vitro DNA primer extension experiments further revealed that F-ara-ATP compared with dATP for incorporation into the A site of the extending DNA strand. The incorporation of F-ara-AMP into DNA resulted in termination of DNA strand elongation. Human DNA polymerase alpha incorporated more F-ara-AMP into DNA than polymerase epsilon (proliferating cell nuclear antigen-independent DNA polymerase delta) and was more sensitive to inhibition by F-ara-ATP. On the other hand, DNA polymerase epsilon was able to excise the incorporated F-ara-AMP from DNA in vitro. The incorporation of F-ara-AMP into DNA was linearly correlated both with inhibition of DNA synthesis and with loss of clonogenicity; thus it may be the mechanism of cytotoxicity.     

Association of 3'----5' exodeoxyribonuclease activity with DNA replitase complex from S-phase Chinese hamster embryo fibroblast cells

DNA replitase has been described as a complex of enzymes/proteins that are associated with both DNA precursor biosynthesis and DNA replication in mammalian cells [Reddy, G. P. V., and Pardee, A. B. (1980) Proc. Natl. Acad. Sci. USA 77, 3312-3316]. We demonstrate for the first time a 3'----5' exodeoxyribonuclease activity is associated with the replitase complex. As much as 60% of this exonuclease activity was similar to that associated with DNA polymerase delta based upon its sensitivity to inhibition by GMP and by butyl-phenyl-deoxyguanosine triphosphate (BuPdGTP). Association of 3'----5' exonuclease activity with the DNA polymerase in the replitase complex was also demonstrated by analyzing dTTP turnover to dTMP in an in vitro DNA polymerase assay system. The DNA polymerase activity in replitase complex exhibited a sensitivity to BuPdGTP which both was similar to that of DNA replication in permeable cells and was intermediate between the BuPdGTP inhibition of purified DNA polymerases alpha and delta. These studies suggest that the replitase complex contains 3'----5' exonuclease activity associated with the DNA polymerase activity responsible for nuclear DNA replication in mammalian cells. Further studies are required to determine if these activities are at least partially attributed to DNA polymerase delta.     

A repeat sequence, GGGTTA, is shared by DNA of human herpesvirus 6 and Marek''s disease virus.

Some regions of the genomes of human B-lymphotrophic virus (HBLV), also designated as human herpesvirus 6, and Marek's disease virus were found to hybridize to each other under moderate to stringent conditions, scoring from 10 to 30% base-pair mismatch. Nucleotide sequence analysis showed that a 6-base-pair repetitive sequence, GGGTTA (DR2), present in the IRS-IRL junction region of the Marek's disease virus genome, was also reiterated in the HBLV genome. The function(s) of such a sequence is unknown, but this is the first report of homology between HBLV and a nonhuman herpesvirus.     

Herpes simplex virus-specified DNA polymerase is the target for the antiviral action of 9-(2-phosphonylmethoxyethyl)adenine.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral compound with activity against herpes simplex virus (HSV) and retroviruses including human immunodeficiency virus. Although it has been suggested that the anti-HSV action of PMEA is through inhibition of the viral DNA polymerase via the diphosphorylated metabolite of PMEA (PMEApp), no conclusive evidence for this has been presented. We report that in cross-resistance studies, a PMEA-resistant HSV variant (PMEAr-1) was resistant to phosphonoformic acid, a compound which directly inhibits the HSV DNA polymerase. In addition, phosphonoformic acid-resistant HSV variants with defined drug resistance mutations within the HSV DNA polymerase gene were resistant to PMEA. Furthermore, the HSV DNA polymerase purified from PMEAr-1 was resistant to PMEApp in comparison with the enzyme from the parental virus. Moreover, PMEA inhibited HSV DNA synthesis in cell culture. These results provide strong evidence that HSV DNA polymerase is the major target for the anti-viral action of PMEA. Further studies showed that HSV DNA polymerase incorporated PMEApp into DNA in vitro, while the HSV polymerase-associated 3'-5' exonuclease was able to remove the incorporated PMEA. Thus, the inhibition of HSV DNA polymerase by PMEApp appears to involve chain termination after its incorporation into DNA.     

Differential inactivation of DNA polymerases α and β by aldehyde compounds

The effects of cyclohexanecarboxaldehyde, benzaldehyde and protocatechualdehyde on the activities of DNA polymerases α, β and E. coli DNA polymerase I were investigated. On direct addition of the aldehydes to the DNA polymerase assay mixture containing activated DNA or poly(dA) (dT)_12–18 as a template, DNA polymerase α was most strongly inhibited by the aldehyde compounds, while DNA polymerases β and I were resistant to such aldehyde inhibition. On preincubation of the enzymes with aldehyde, both DNA polymerases α and β were inactivated; however, DNA polymerase β was protected from the inactivation when activated DNA was added to the preincubation mixture. The inhibition of DNA polymerase α by aldehyde was noncompetitive with regard to the substrate dNTP and competitive with regard to the template DNA. The extent of inhibition of DNA polymerase α by aldehyde was partly reduced by the addition of cysteine to the reaction mixture.     

The inhibition of mitochondrial DNA polymerase gamma from animal cells by intercalating drugs.

DNA polymerase gamma from purified nuclei of EMT-6 cells (mice) seems to be identical to the mitochondrial DNA polymerase from the same source following several criteria. These two enzyme activities are strongly inhibited by ethidium bromide and acriflavin, while proflavin, acridine orange, daunomycin and chloroquine inhibition is less pronounced. In the case of DNA polymerases alpha and beta very little inhibition by ethidium bromide was observed. Intercalation of this dye in a poly dA-dT 12-18 template-primer was studied spectrophotometrically under conditions similar to those in the in vitro DNA polymerase assay. The polymerase assay. The inhibition by this drug of the mitochondrial DNA polymerase gamma activity was shown to be competitive at varying concentrations of TTP while the inhibition was of the non-competitive type at different concentrations of poly dA-dT 12-18. We conclude that the drug, most probably in the intercalated form, is able to interact with the active site (s) of mitochondrial DNA polymerase.     

Mechanism of inhibition of RNA polymerase II and poly(adenylic acid) polymerase by the O-n-octyloxime of 3-formylrifamycin SV.

Factors affecting the inhibition of RNA polymerase II from rat liver by the O-n-octyloxime of 3-formylrifamycin SV (AF/013) were investigated. Using either native or denatured calf-thymus DNA as template, almost complete inhibition of RNA polymerase II was observed when AF/013 was added directly to the enzyme. Considerable resistance to AF/013 was observed when RNA polymerase II was preincubated with denatured DNA at either 0 or 37 degrees. However, under similar conditions, no resistance was observed when enzyme was preincubated with native DNA. Only when AF/013 was added to the ongoing reaction using native DNA did a resistance to AF/013 occur. The inhibition of RNA polymerase II by AF/013 was competitive with respect to all four nucleoside triphosphate substrates. The inhibition by AF/013 remaining after enzyme-DNA complex formation also appeared competitive with nucleoside triphosphate levels. The effect of exogenous protein (bovine serum albumin, BSA) on the inhibition of RNA polymerase II was also investigated. BSA reduced the extent of inhibition by AF/013, but did not alter the competitive nature of inhibition. Concurrently, the inhibition of highly purified nuclear poly(A) polymerase from rat liver, a template independent enzyme which incorporates AMP in a chain elongation reaction, was examined. As in the case of RNA polymerase, poly(A) polymerase was inhibited by AF/013 in a manner competitive with the nucleoside triphosphate substrate. The competitive nature of inhibition of RNA polymerase by AF/013 with respect to all four nucleoside triphosphate substrates, before and after enzyme-DNA complex formation, as well as the competitive nature of inhibition of poly(A) polymerase with respect to ATP tend to indicate that the major effect of AF/013 on RNA polymerase II is at the level of the substrate binding as opposed to a specific inhibition of initiation.     

Comparative effects of the 5'-triphosphates of 9-beta-(2'-azido-2'-deoxy-D-arabinofuranosyl)adenine and 9-beta-D-arabinofuranosyladenine on DNA polymerases from L1210 leukemia cells.

9-beta-(2'-Azido-2'-deoxy-D-arabiofuranosyl)adenine (arazide) causes greater and significantly more persistent inhibition of [3H]-thymidine incorporation into the DNA of neoplastic cells than the related agent 9-beta-D-arabinofuranosyladenine (araA). To elucidate the mechanism(s) responsible, we compared the effects of arazide and araA 5'-triphosphates on DNA polymerases alpha and beta of L1210 leukemia cells. Both nucleoside triphosphate analogs inhibited DNA polymerase alpha activity by competing with dATP; only araATP was inhibitory to DNA polymerase beta. Arazide triphosphate was at least four times more active than araATP as an inhibitor of DNA polymerase alpha. Preincubation of DNA polymerase alpha with either agent did not result in enzyme inactivation. The results suggest that interference with DNA polymerase alpha activity by arazide triphosphate may be in part responsible for the inhibition of DNA synthesis produced by arazide in neoplastic cells.     

Inhibition of DNA polymerase by captan

Captan inhibits DNA polymerases of both eukaryotic and prokaryotic sources. When polymerases were employed in assays with various polynucleotides as template-primer, no specificity in the base sequence of polynucleotide was required for inhibition. Sucrose gradient centrifugation and preincubation studies showed the inhibition was caused by an irreversible alteration of the polymerase. Captan and DNA compete for the same site on the polymerase, thus DNA can serve a protective role in the elimination of captan's action. The pyrophosphate exchange activity associated with the polymerase is not inhibited by captan and the fidelity with which DNA polymerase I copies the DNA template also is not altered by captan treatment.     

Mechanistic Independence of Avian Myeloblastosis Virus DNA Polymerase and Ribonuclease H

Differential inhibition conditions were established for the DNA polymerase and RNase H activities of avian myeloblastosis virus (AMV) with ether-disrupted AMV and a purified enzyme preparation. The RNase H activity of ether-disrupted AMV with (rA)(n).(dT)(n) and (rA)(n).(dT)(11) as substrates was inhibited 80 to 100% by preincubation with NaF at a final reaction concentration of 27 to 30 mM. Under these conditions, the DNA polymerase activity was inhibited only 0 to 20%. Similar inhibitions were found with exogenous Rous sarcoma virus 35S and 70S RNA.DNA hybrid and phiX174 DNA.RNA hybrid as substrates. Studies were also performed with a purified enzyme preparation, in which the two activities essentially co-purified. The RNase H activity was inhibited >80% by 150 mM KCl with three different hybrid substrates, whereas the DNA polymerase activity was uninhibited. The DNA polymerase was completely inactivated by heat denaturation at 41 C or by omission of the deoxytriphosphates from the reaction mixture; the RNase H remained active. These differential inhibition conditions were used to compare the size of the DNA product synthesized with and without simultaneous RNase H action and to examine the effect of inhibition of the DNA polymerase on the size of the RNase H products. The size of the products of one activity was not affected by inhibition of the other activity. These results suggest that the AMV DNA polymerase and RNase H are not coupled mechanistically.     

Mechanism of inhibition of human cytomegalovirus replication by oxetanocin G.

Oxetanocin G(9-(2-deoxy-2-hydroxymethyl-beta-D-erythro-oxetanosyl)guanine, OXT-G) is a potent and selective agent against human cytomegalovirus (HCMV). In this study we synthesized the triphosphate form of OXT-G, OXT-GTP, and examined its effect on the activities of HCMV DNA polymerase, herpes simplex type 2 (HSV-2) DNA polymerase and human DNA polymerase alpha. OXT-GTP was found to inhibit all these polymerases in a competitive manner with respect to dGTP. The Km for dGTP and the Ki for OXT-GTP of HCMV DNA polymerase were 0.86 and 0.53 mu M, respectively, while the corresponding values of DNA polymerase alpha were 2.2 and 3.6 mu M, respectively. HPLC analysis using [3H]OXT-G also revealed that OXT-G was converted to its triphosphate form 7- to 8-fold more efficiently in HCMV-infected cells than in uninfected cells. The results suggest that both the preferential phosphorylation of OXT-G in HCMV-infected cells and the preferential inhibition of HCMV DNA polymerase by OXT-GTP may contribute towards the selective activity of OXT-G against HCMV replication.     

Template properties of DNA alkylated with N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea

Alkylation of DNA with N-methyl-N-nitrosourea (MeNU) and N-ethyl-N-nitrosourea (EtNU) reduces its ability to support RNA synthesis catalyzed by exogenously added RNA polymerase. It is likely that 7-alkylguanine and alkyl phosphotriester in DNA are mainly responsible for the inhibition of RNA synthesis. The inhibitory effect of alkyl groups varies depending upon divalent metal ions and the type of RNA polymerase used as well as upon the presence of chromosomal proteins on DNA templates. Analyses of RNA products indicate that inhibition occurs primarily at the initiation step.     

Nalidixic Acid and the Metabolism of Escherichia coli

Nalidixic acid (NAL) is bactericidal for E. coli B. Synthesis of deoxyribonucleic acid (DNA), ribonucleic acid and protein was necessary to initiate the lethal effect, but only protein synthesis was necessary to sustain it. NAL inhibited DNA synthesis specifically, but this inhibition occurred even under conditions that were not lethal to the bacteria. In contrast to other inhibitors of DNA synthesis, NAL did not cause the solubilization of cellular DNA even when bacteria were exposed to it for 2 hr. A bacterial mutant deficient in DNA polymerase was much more sensitive to the lethal action of NAL than its parent strain. Moreover, inhibition of protein synthesis did not protect this mutant from NAL-induced killing. NAL inhibited neither DNA polymerase, nor thymidine or thymidylate kinases. The data are interpretated as suggesting that NAL altered the structure of DNA or a protein attached to nascent DNA and that this lesion can be partially repaired by DNA polymerase.     

Kinetics of the decamer - dimer dissociation of arginine decarboxylase.

Aurintricarboxylic acid inhibited replicative DNA synthesis in nucleotide-permeable mouse ascites sarcoma cells. DNA polymerase activity assayed with activated DNA template and DNA polymerase purified partially from sarcoma cells was also inhibited by aurintricarboxylic acid. The inhibition of DNA polymerase activity was probably due to the inhibitory interaction of aurintricarboxylic acid with DNA polymerase. The replicative DNA synthesis might be inhibited by aurintricarboxylic acid interacting with some essential protein component(s), such as DNA polymerase of the replication machinery.