Shonen Yoshida

FATTY ACIDS SELECTIVELY INHIBIT EUKARYOTIC DNA POLYMERASE ACTIVITIES IN VITRO

The in vitro relationship between eukaryotic DNA polymerases and fatty acids was investigated. Some fatty acids strongly inhibited the activities of DNA polymerase alpha and/or beta in vitro. The kinetics of inhibition by linoleic acid showed that DNA polymerase alpha was non-competitively inhibited with respect to the DNA template and substrate (dTTP), while DNA polymerase beta was inhibited competitively with both DNA and substrate.

The inhibitory action of fatty acids on DNA polymerase β

We found previously that long-chain fatty acids could inhibit eukaryotic DNA polymerase activities in vitro [1,2]. The purpose of the present study was to investigate the mode of this inhibition in greater detail. Among the C18 to C24 fatty acids examined, the strongest inhibitor was a C24 fatty acid, nervonic acid (NA), and the weakest was a C18 fatty acid, linoleic acid (LA). We analyzed the inhibitory effect of these two fatty acids and their modes of action. For DNA polymerase beta (pol. beta), NA acted by competing with both the substrate- and template-primer, but for DNA polymerase alpha (pol. alpha) or human immunodeficiency virus type 1 reverse transcriptase (HIV-1 reverse transcriptase or HIV-RT), NA acted non-competitively. NA-binding to pol. beta could be stopped with a non-ionic detergent, but the binding to pol. alpha or HIV-RT could not. The inhibition mode of LA showed the same characteristics, except that the minimum inhibitory dose of the longer chain was much lower. We also tested the effects of NA and LA using pol. beta and its proteolytic fragments, as described by Kumar et al. [3,4]. Both of the fatty acids were found to bind to the 8 kDa DNA-binding domain fragment, and to suppress binding to the template-primer DNA. We found that 10,000 times more of either fatty acid was required for it to bind to the 31 kDa catalytic domain or inhibit the DNA polymerase activity. The possible modes of inhibition by these long-chain fatty acids are discussed, based on the present findings.

Structural study of immunoaffinity-purified DNA polymerase α-DNA primase complex from calf thymus

The DNA polymerase alpha-DNA primase complex was purified over 17,000-fold to near homogeneity from calf thymus using an immunoaffinity column. Sodium dodecyl sulfate gel electrophoresis revealed three polypeptides with molecular weights of 140, 50 and 47 kDa, in a ratio of 1:2:0.25. The complex showed a sedimentation coefficient of 9.7 S, a Stokes radius of 56 A and a native molecular weight of 250-260 kDa. Taken together, the data suggest that the calf thymus dNA polymerase alpha-DNA primase complex is essentially a heterotrimer of large (140 kDa) and small (50 kDa) subunits in a ratio of 1:2, with a globular conformation. Electron-microscopic studies of the complex revealed a spherical particle of 120 A in diameter, in agreement with the physiochemical results. The binding of the complex to DNA was also demonstrated.

Intracellular localization and metabolism of DNA polymerase α in human cells visualized with monoclonal antibody

Indirect immunofluorescence microscopy with monoclonal antibody against DNA polymerase alpha revealed the intranuclear localization of DNA polymerase alpha in G1, S, and G2 phases of transformed human cells, and dispersed cytoplasmic distribution during mitosis. In the quiescent, G0 phase of normal human skin fibroblasts or lymphocytes, the alpha-enzyme was barely detectable by either immunofluorescence or enzyme activity. By exposing cells to proliferation stimuli, however, DNA polymerase alpha appeared in the nuclei just prior to onset of DNA synthesis, increased rapidly during S phase, reached the maximum level at late S and G2 phases, and was then redistributed to the daughter cells through mitosis. It was also found that the increase in the amount of DNA polymerase alpha by proliferation stimuli was not affected by inhibition of DNA synthesis with aphidicolin or hydroxyurea.

Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

ABSTRACT We isolated active mutants in Saccharomyces cerevisiae DNA polymerase α that were associated with a defect in error discrimination. Among them, L868F DNA polymerase α has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase α. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase α-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase α catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3′ T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase η, and the F34L mutant of S. cerevisiae DNA polymerase η has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase α is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.

Lithocholic acid, a putative tumor promoter, inhibits mammalian DNA polymerase β

Lithocholic acid (LCA), one of the major components in secondary bile acids, promotes carcinogenesis in rat colon epithelial cells induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), which methylates DNA. Base‐excision repair of DNA lesions caused by the DNA methylating agents requires DNA polymerase β (pol β). In the present study, we examined 17 kinds of bile acids with respect to inhibition of mammalian DNA polymerases in vitro. Among them, only LCA and its derivatives inhibited DNA polymerases, while other bile acids were not inhibitory. Among eukaryotic DNA polymerases α, β, δ, η, and γ, pol β was the most sensitive to inhibition by LCA. The inhibition mode of pol β was non‐competitive with respect to the DNA template‐primer and was competitive with the substrate, dTTP, with the Ki value of 10 μM. Chemical structures at the C‐7 and C‐12 positions in the sterol skeleton are important for the inhibitory activity of LCA. This inhibition could contribute to the tumor‐promoting activity of LCA.

Studies on inhibitors of mammalian DNA polymerase α and β: Sulfolipids from a pteridophyte, Athyrium niponicum

Abstract Three sulfolipid compounds, 1, 2 and 3 , have been isolated from a higher plant, a pteridophyte, Athyrium niponicum , as potent inhibitors of the activities of calf DNA polymerase α and rat DNA polymerase β. The inhibition by the sulfolipids was concentration dependent, and almost complete inhibition of DNA polymerase α and DNA polymerase β was achieved at 6 and 8 μg/mL, respectively. The compounds did not influence the activities of calf thymus terminal deoxynucleotidyl transferase, prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase and Taq polymerase, the DNA metabolic enzyme DNase I, and even a DNA polymerase from a higher plant, cauliflower. Similarly, the compounds did not inhibit the activity of the human immunodeficiency virus type 1 reverse transcriptase. The kinetic studies of the compounds showed that DNA polymerase α was inhibited non-competitively with respect to the DNA template and substrate, whereas DNA polymerase β was inhibited competitively with both the DNA template and substrate. The binding to DNA polymerase β could be stopped with non-ionic detergent, but the binding to DNA polymerase α could not.

Multiple molecular species of cytoplasmic DNA polymerase from calf thymus

Abstract Cytoplasmic DNA polymerase preparation from calf thymus was separated into three peaks on DEAE-cellulose column. All these three species preferred activated DNA to denatured DNA and were distinguished by their template specificities, isoelectric points, and the sedimentation rates on the sucrose gradients. Sedimentation rates of these three species lay between 6.7 and 8.5 ( M r 130 000–200 000). These three species were further dissociated into active smaller molecules ( M r 90 000 and 50 000–60 000) under certain conditions, such as a treatment with 2 M urea. Small active molecular weight species were also obtained by gel filtration on Sephadex G-200. These results suggest the existence of small active subunits in large DNA polymerase species in the cytoplasm of calf thymus.

Inhibition of DNA polymerase-α and -β of calf thymus by 1-β-d-arabinofuranosylcytosine-5′-triphosphate

Abstract 1-β- d -Arabinofuranosylcytosine 5′-triphosphate (araCTP), an active form of a inhibitor of DNA replication, 1-β- d -arabinofuranosylcytosine (araC) was tested for its inhibitory action on the DNA polymerase-α and -β (EC 2.7.7.7) purified from calf thymus. The reaction of DNA polymerase-α was shown to be more sensitive to the inhibition by araCTP than that of DNA polymerase-β. The mode of the inhibition by araCTP was competitive to dCTP in the reaction catalysed by either DNA polymerase-α or -β. The K i value of DNA polymerase-β for araCTP was 32 μM; eight times higher than that of DNA polymerase-α (4 μM) for this inhibitor.

A possible role of nuclear ceramide and sphingosine in hepatocyte apoptosis in rat liver

BACKGROUND/AIMS Portal vein branch ligation induces apoptosis of hepatocytes in the ligated lobes in rat liver. Sphingomyelin degradation was studied during the process to evaluate its possible involvement in apoptosis in vivo. METHODS DNA scissions were detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and an agarose gel electrophoresis of DNA. Using both ligated and non-ligated lobes, we measured activities of sphingomyelin degradation enzymes and contents of their products in purified nuclei and plasma membrane. RESULTS DNA fragmentation was detectable in the ligated lobes at 90 min after the portal vein branch ligation by gel electrophoresis. At 15 h after the ligation, 27% of hepatocytes became TUNEL-positive. Prior to the onset of apoptosis, the activity of neutral sphingomyelinase increased in the nuclei of hepatocytes in ligated lobes (30 min after the ligation). The increase in sphingomyelinase paralleled its reaction product, ceramide. This was followed by the elevation of ceramidase activity in nuclei (60 min after the ligation) in association with an increase of its reaction product, sphingosine. Activities of these two enzymes and their products increased for at least 90 min. These changes were not observed in nuclei of the non-ligated lobes, or in the plasma membranes from either ligated or non-ligated lobes. CONCLUSIONS These results, specific to the liver where apoptosis is being generated, suggest that nuclear sphingomyelin breakdown with an accumulation of ceramide and/or sphingosine in nuclei may induce the apoptosis of hepatocytes in vivo.