Eukaryotic DNA Polymerases Research Articles

A heterodimeric DNA polymerase: evidence that members of Euryarchaeota possess a distinct DNA polymerase.

We describe here a DNA polymerase family highly conserved in Euryarchaeota, a subdomain of Archaea. The DNA polymerase is composed of two proteins, DP1 and DP2. Sequence analysis showed that considerable similarity exists between DP1 and the second subunit of eukaryotic DNA polymerase delta, a protein essential for the propagation of Eukarya, and that DP2 has conserved motifs found in proteins with nucleotide-polymerizing activity. These results, together with our previous biochemical analyses of one of the members, DNA polymerase II (DP1 + DP2) from Pyrococcus furiosus, implicate the DNA polymerases of this family in the DNA replication process of Euryarchaeota. The discovery of this DNA-polymerase family, aside from providing an opportunity to enhance our knowledge of the evolution of DNA polymerases, is a significant step toward the complete understanding of DNA replication across the three domains of life.

Lithocholic acid, a putative tumor promoter, inhibits mammalian DNA polymerase beta.

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.

Overexpression of DNA polymerase beta in cell results in a mutator phenotype and a decreased sensitivity to anticancer drugs.

DNA polymerase beta (pol beta) is the most error prone of all known eukaryotic DNA polymerases tested in vitro. Here, we show that cells overexpressing pol beta cDNA have acquired a spontaneous mutator phenotype. By measuring the appearance of mutational events using three independent assays, we found that genetic instability increased in the cell lines that overexpressed pol beta. In addition, these cells displayed a decreased sensitivity to cancer chemotherapeutic, bifunctional, DNA-damaging agents such as cisplatin, melphalan, and mechlorethamine, resulting in enhanced mutagenesis compared with control cells. By using cell-free extracts and modified DNA substrates, we present data in support of error-prone translesion replication as one of the key determinants of tolerance phenotype. These results have implications for the potential role of pol beta overexpression in cancer predisposition and tumor progression during chemotherapy.

Eukaryotic DNA polymerases in DNA replication and DNA repair.

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair. Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA polymerases in these different pathways. DNA polymerase alpha is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme. DNA polymerase delta functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase zeta, for mutagenesis. The function of DNA polymerase epsilon in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase delta or epsilon suffices for the repair of UV-induced damage. The role of DNA polymerase beta in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase delta appears to fulfill that function.

Effects of Aphidicolin on Entamoeba histolytica Growth and DNA Synthesis

We have previously demonstrated that DNA polymerase activity of Entamoeba histolytica is inhibited by aphidicolin, which is a specific inhibitor of eukaryotic nuclear replicative DNA polymerases. The present study was aimed to evaluate the effect of aphidicolin on growth and DNA synthesis by this parasite. Aphidicolin blocked the growth of axenic E. histolytica strain HM-1:IMSS. DNA synthesis was also inhibited by aphidicolin when assayed by incorporation of [3H]thymidine into the DNA. The inhibitory effect of aphidicolin on the growth of E. histolytica was abrogated by removal of the drug, and exposure to 3 microg/ml of the drug for at least 48 hr had little effect on the viability. Synchronous growth was observed in the recovery phase after removal of aphidicolin.

A Mushroom Fruiting Body-Inducing Substance Inhibits Activities of Replicative DNA Polymerases

We found and isolated two natural products in the extract from a basidiomycete,Ganoderma lucidum,as eukaryotic DNA polymerase inhibitors. The compounds were identified as cerebrosides, (4E,8E)-ND-2′-hydroxypalmitoyl-1-O-β-D-glucopyranosyl-9methyl-4,8-sphingadienine and (4E,8E)-N-D-2′-hydroxystearoyl-1-O-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine, and were found to be identical to the mushroom fruiting body-inducing substances (FIS) reported. These cerebrosides selectively inhibited the activities of replicative DNA polymerases, especially the α-type, from phylogenetically broad eukaryotic species, whereas they hardly influenced the activities of DNA polymerase β, prokaryotic DNA polymerases, terminal deoxynucleotidyl transferase, HIV reverse transcriptase, RNA polymerase, deoxyribonuclease I, and ATPase. The inhibition of another replicative polymerase, the δ-type, was moderate. The inhibitions of the replicative polymerases were dose-dependent, and the IC50for animal or mushroom DNA polymerase α was achieved at approximately 12 μg/ml (16.2 μM) and for animal DNA polymerase δ at 57 μg/ml (77.2 μM). FIS is possibly a DNA polymerase inhibitor specific to the replicative enzyme group, and the fruiting body formation may be required for the suppression of the DNA replication or the vegetative growth of the mycelium.

A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase zeta.

To get a better understanding of mutagenic mechanisms in humans, we have cloned and sequenced the human homolog of the Saccharomyces cerevisiae REV3 gene. The yeast gene encodes the catalytic subunit of DNA polymerase zeta, a nonessential enzyme that is thought to carry out translesion replication and is responsible for virtually all DNA damage-induced mutagenesis and the majority of spontaneous mutagenesis. The human gene encodes an expected protein of 3,130 residues, about twice the size of the yeast protein (1,504 aa). The two proteins are 29% identical in an amino-terminal region of approximately 340 residues, 39% identical in a carboxyl-terminal region of approximately 850 residues, and 29% identical in a 55-residue region in the middle of the two genes. The sequence of the expected protein strongly predicts that it is the catalytic subunit of a DNA polymerase of the pol zeta type; the carboxyl-terminal domain possesses, in the right order, the six motifs characteristic of eukaryotic DNA polymerases, most closely resembles yeast pol zeta among all polymerases in the GenBank database, and is different from the human alpha, delta, and epsilon enzymes. Human cells expressing high levels of an hsREV3 antisense RNA fragment grow normally, but show little or no UV-induced mutagenesis and are slightly more sensitive to killing by UV. The human gene therefore appears to carry out a function similar to that of its yeast counterpart.

Sulfated glycoglycerolipid from archaebacterium inhibits eukaryotic DNA polymerase α, β and retroviral reverse transcriptase and affects methyl methanesulfonate cytotoxicity

A sulfated glycoglycerolipid, 1-O-(6′-sulfo-α-d-glucopyranosyl)-2,3-di-O-phytanyl-sn-glycerol (KN-208), a derivative of the polar lipid isolated from an archaebacterium, strongly inhibited DNA polymerase (pol) α and pol β in vitro among 5 eukaryotic DNA polymerases (α, β, γ, δ, and ϵ). It also inhibited Escherichia coli DNA polymerase I Klenow fragment (E. coli pol I) and human immunodeficiency virus reverse transcriptase (HIV RT). The mode of inhibition of these polymerases was competitive with the DNA template primer and was non-competitive with the substrate dTTP. KN-208 inhibited pol β most strongly, with a Ki value of 0.05 μM, 10-fold lower than that for pol α (0.5 μM) and 60- or 140-fold lower than that for HIV RT (3 μM) or for E. coli pol I (7 μM), respectively. The loss of sulfate on the 6′-position of glucopyranoside of this compound completely abrogated inhibition. However, the hydrophilic part of KN-208, glucose 6-sulfate alone, showed no inhibition. Other sulfated compounds containing different hydrophobic structures, such as dodecyl sulfate and cholesterol sulfate, exhibited a much weaker inhibition. Our results suggest that the whole molecular structure of KN-208 is required for inhibition. KN-208 was shown to be modestly cytotoxic for the human leukemic cell line K562. Interestingly, a subcytotoxic dose of KN-208 increased the sensitivity of the human leukemic cells to an alkylating agent, methyl methanesulfonate, while it did not potentiate the effects of ultraviolet light or of cisplatin. Int. J. Cancer 76:512–518, 1998.© 1998 Wiley-Liss, Inc.

Sulfated glycoglycerolipid from archaebacterium inhibits eukaryotic DNA polymerase alpha, beta and retroviral reverse transcriptase and affects methyl methanesulfonate cytotoxicity.

A sulfated glycoglycerolipid, 1-O-(6'-sulfo-alpha-D-glucopyranosyl)-2,3-di-O-phytanyl- sn-glycerol (KN-208), a derivative of the polar lipid isolated from an archaebacterium, strongly inhibited DNA polymerase (pol) alpha and pol beta in vitro among 5 eukaryotic DNA polymerases (alpha, beta, gamma, delta, and epsilon). It also inhibited Escherichia coli DNA polymerase I Klenow fragment (E. coli pol I) and human immunodeficiency virus reverse transcriptase (HIV RT). The mode of inhibition of these polymerases was competitive with the DNA template primer and was non-competitive with the substrate dTTP. KN-208 inhibited pol beta most strongly, with a Ki value of 0.05 microM, 10-fold lower than that for pol alpha (0.5 microM) and 60- or 140-fold lower than that for HIV RT (3 microM) or for E. coli pol I (7 microM), respectively. The loss of sulfate on the 6'-position of glucopyranoside of this compound completely abrogated inhibition. However, the hydrophilic part of KN-208, glucose 6-sulfate alone, showed no inhibition. Other sulfated compounds containing different hydrophobic structures, such as dodecyl sulfate and cholesterol sulfate, exhibited a much weaker inhibition. Our results suggest that the whole molecular structure of KN-208 is required for inhibition. KN-208 was shown to be modestly cytotoxic for the human leukemic cell line K562. Interestingly, a subcytotoxic dose of KN-208 increased the sensitivity of the human leukemic cells to an alkylating agent, methyl methanesulfonate, while it did not potentiate the effects of ultraviolet light or of cisplatin.

Recognition of 2'-deoxyisoguanosine triphosphate by HIV-1 reverse transcriptase and mammalian cellular DNA polymerases.

HIV-1 reverse transcriptase (RT) incorporates 2'-deoxyisoguanosine triphosphate (d-isoGTP) opposite thymidine (T) in a DNA template and opposite uracil (U) in an RNA template about 10 times more efficiently than the eukaryotic DNA polymerase alpha, both in the absence and presence of dATP.

The specific inhibitory effect of demethoxydehydroaltenusin, a derivative of dehydroaltenusin, on mammalian DNA polymerase α

In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin from the fungus Acremonium sp. was found to be an inhibitor of pol alpha. The present study succeeded in chemically synthesizing dehydroaltenusin, and the compound strongly inhibited calf pol alpha activity and weakly suppressed rat pol beta activity, with IC50 values of 0.68 and 64 microM, respectively. We purified or synthesized various slightly modified derivatives of dehydroaltenusin, and using these, investigated the relationship between chemical structure and the inhibitory effects. These results suggest that the ketone group at the 5'-position in dehydroaltenusin is essential for pol inhibitory activity, and the group at the 5-position is important for the specificity of pol alpha inhibition. Demethoxydehydroaltenusin was found to be the most specific pol alpha inhibitor among the prepared derivatives, and the IC50 values for pols alpha and beta were 0.24 and 89 microM, respectively. This compound did not influence the activities of other replicative pols such as pols delta and epsilon, and also demonstrated no effect on pol alpha activity from another vertebrate, fish and a plant species. Demethoxydehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. Therefore, demethoxydehydroaltenusin is of interest as a mammalian pol alpha-selective inhibitor as a 'chemical knockout agent' in vitro and in vivo.

Synthetic Nucleosides and Nucleotides. 40. Selective Inhibition of Eukaryotic DNA Polymerase α by 9-(β-D-Arabinofuranosyl)-2-(p-n-butylanilino)adenine 5′-Triphosphate (BuAaraATP) and Its 2′-Up Azido Analog: Synthesis and Enzymatic Evaluations†

Starting from 2′,3′,5′-tri-O-acetyl-2-iodoadenosine, 9-(β-D-arabinofuranosyl)-2-(p-n-butylanilino)adenine and its 2′(S)-azido counterparts were synthesized in seven steps. These exhibited only moderate growth-inhibitory effects against mouse leukemic P388 cells (IC50 = 13–24 μM), although 5′-triphosphate derivatives showed strong and selective inhibitory action on calf thymus DNA polymerase α, but not on β- and ϵ-polymerases from eukaryotes. This paper is dedicated to the memory of Professor Tsujiaki Hata

Sulfoquinovosyldiacylglycerol, KM043, a new potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1 from a marine red alga, Gigartina tenella.

A new sulfolipid, KM043, which belongs to the 6-sulfo-alpha-D-quinovopyranosyl-(1-->3')-1',2'-diacylglycerol (SQDG) class of compounds, has been isolated from a marine red alga, Gigartina tenella, as a potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1. Its structure was determined on the basis of spectroscopic and gas chromatographic analyses. The inhibition was dose-dependent, and complete (more than 90%) inhibition of DNA polymerase alpha (pol. alpha), DNA polymerase beta (pol. beta) and HIV-reverse transcriptase type 1 (HIV-RT) was observed at concentrations of 5, 10, and 30 microM, respectively.

Inhibitory effects of vitamin K3 on DNA polymerase and angiogenesis

Vitamins play essential roles in cellular reactions and maintain human health. Recent studies have revealed that some vitamins including D3, B6 and K2 and their derivatives have an anti-cancer effect. As a mechanism, their inhibitory effect on cancer-related angiogenesis has been demonstrated. Vitamin K2 (menaquinones) has an anti-cancer effect in particular for hepatic cancer and inhibits angiogenesis. In the current study, we demonstrated that sole vitamin K3 (menadione) selectively inhibits the in vitro activity of eukaryotic DNA polymerase gamma, which is a mitochondrial DNA polymerase, and suppresses angiogenesis in a rat aortic ring model. The anti-angiogenic effect of vitamin K3 has been shown in angiogenesis models using human umbilical vein endothelial cells (HUVECs) with regard to HUVEC growth, tube formation on reconstituted basement membrane and chemotaxis. These results suggest that vitamin K3 may be a potential anti-cancer agent like vitamin K2.

Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.

The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.

The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis.

Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.

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 C 18 to C 24 fatty acids examined, the strongest inhibitor was a C 24 fatty acid, nervonic acid (NA), and the weakest was a C 18 fatty acid, linoleic acid (LA). We analyzed the inhibitory effect of these two fatty acids and their modes of action. For DNA polymerase β (pol. β), NA acted by competing with both the substrate- and template-primer, but for DNA polymerase α (pol. α) or human immunodeficiency virus type 1 reverse transcriptase (HIV-1 reverse transcriptase or HIV-RT), NA acted non-competitively. NA-binding to pol. β could be stopped with a non-ionic detergent, but the binding to pol. α 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. β 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.

Phylogenetic relationships among hypotrichous ciliates determined with the macronuclear gene encoding the large, catalytic subunit of DNA polymerase alpha.

The complete macronuclear DNA polymerase alpha gene, previously sequenced in Oxytricha nova, has been cloned from a genomic macronuclear library and sequenced for the hypotrich O. trifallax. Macronuclear DNA clones of DNA polymerase alpha encoding approximately 1000 amino acids, or approximately two-thirds of the open reading frame, have been obtained by PCR and sequenced for Halteria grandinella, Holosticha species, Paraurostyla viridis, Pleurotricha lanceolata, Stylonychia lemnae Teller, Sty. mytilus, Uroleptus gallina, and Urostyla grandis. Phylogenetic relationships inferred from DNA polymerase alpha amino acid sequences have been used to clarify taxonomic relationships previously determined by morphology of the cell cortex. Hypotrich phylogenies based on DNA polymerase alpha amino acid sequences are incongruent with morphological and other molecular phylogenies. Based upon these data, we assert that, contrary to morphological data, O. nova and O. trifallax are different species, and we propose that the oligotrich Halteria grandinella be reclassified as a hypotrich. This work also extends the available data base of eukaryotic DNA polymerase alpha sequences, and suggests new amino acid sequence targets for mutagenesis experiments to continue the functional dissection of DNA pol alpha biochemistry at the molecular level.

A novel DNA polymerase in the hyperthermophilic archaeon, Pyrococcus furiosus: gene cloning, expression, and characterization.

In many respects Archaea are much more like eukaryotes than prokaryotes with respect to the conservation of many of the components involved in transcription, translation and DNA replication. So far, only a few DNA polymerases with structures similar to those of eukaryotic DNA polymerase a have been found in Archaea. The identification and characterization of all the DNA polymerases of one archaeon would add considerably to our knowledge of the basic mechanisms of DNA replication in these organisms. We have identified a novel DNA polymerase composed of two proteins, DP1 and DP2, with molecular weights of 69294 Da and 143161 Da, respectively, in the hyperthermophilic archaeon, Pyrococcus furiosus, and have cloned the corresponding genes which are tandemly arranged on the Pyrococcus genome. No significant sequence homology was found between these two proteins and other known DNA polymerases. The pol genes were transcribed as part of a single operon that additionally contained genes homologous to the cdc18+/CDC6 and Dmc1/Rad51 family of proteins. We purified the Pyrococcus DNA polymerase from Escherichia coli strains expressing the cloned genes and characterized its activity. It possesses strong 3' --> 5' exonucleolytic activity and has a template-primer preference which is characteristic of a replicative DNA polymerase. In P. furiosus, we identified a second DNA polymerase encoded by two genes, neither of which display significant homology to any other known DNA polymerase. Both the enzymatic properties of the enzyme and the gene organization raise the possibility that this enzyme might be the replicative DNA polymerase of P. furiosus.

Crystal Structure of a pol α Family Replication DNA Polymerase from Bacteriophage RB69

The 2.8 Å resolution crystal structure of the bacteriophage RB69 gp43, a member of the eukaryotic pol α family of replicative DNA polymerases, shares some similarities with other polymerases but shows many differences. Although its palm domain has the same topology as other polymerases, except rat DNA polymerase β, one of the three carboxylates required for nucleotidyl transfer is located on a different β strand. The structures of the fingers and thumb domains are unrelated to all other known polymerase structures. The editing 3′–5′ exonuclease domain of gp43 is homologous to that of E. coli DNA polymerase I but lies on the opposite side of the polymerase active site. An extended structure-based alignment of eukaryotic DNA polymerase sequences provides structural insights that should be applicable to most eukaryotic DNA polymerases.