Mammalian DNA Polymerase Research Articles

Structure and function of 2:1 DNA polymerase·DNA complexes

DNA polymerases are required for DNA replication and DNA repair in all of the living organisms. Different DNA polymerases are responsible different stages of DNA metabolism, and many of them are multifunctional enzymes. It was generally assumed that the different reactions are catalyzed by the same enzyme molecule. In addition to 1:1 DNA polymerase.DNA complex reported by crystallization studies, 2:1 and higher order DNA polymerase.DNA complexes have been identified in solution studies by various biochemical and biophysical approaches. Further, abundant evidences for the DNA polymerase-DNA interactions in several DNA polymerases suggested that the 2:1 complex represents the more active form. This review describes the current status of this emerging subject and explores their potential in vitro and in vivo functional significance, particularly for the 2:1 complexes of mammalian DNA polymerase beta (Pol beta), the Klenow fragment of E. coli DNA polymerase I (KF), and T4 DNA polymerase.

Mismatched dNTP incorporation by DNA polymerase β does not proceed via globally different conformational pathways†

Understanding how DNA polymerases control fidelity requires elucidation of the mechanisms of matched and mismatched dNTP incorporations. Little is known about the latter because mismatched complexes do not crystallize readily. In this report, we employed small-angle X-ray scattering (SAXS) and structural modeling to probe the conformations of different intermediate states of mammalian DNA polymerase β (Pol β) in its wild-type and an error-prone variant, I260Q. Our structural results indicate that the mismatched ternary complex lies in-between the open and the closed forms, but more closely resembles the open form for WT and the closed form for I260Q. On the basis of molecular modeling, this over-stabilization of mismatched ternary complex of I260Q is likely caused by formation of a hydrogen bonding network between the side chains of Gln260, Tyr296, Glu295 and Arg258, freeing up Asp192 to coordinate MgdNTP. These results argue against recent reports suggesting that mismatched dNTP incorporations follow a conformational path distinctly different from that of matched dNTP incorporation, or that its conformational closing is a major contributor to fidelity.

New Cancer Chemotherapy Agents: Inhibitors of DNA Polymerase

The purpose of this review is 1) to introduce our studies of newly-discovered mammalian DNA polymerase inhibitors; 2) to elucidate their precise molecular action based on three-dimensional structural models and comparisons with the spatial positioning on the smallest DNA polymerase, β, of specific amino acids binding to them; and 3) to document their promising clinical anti-tumor activity. Multiple DNA polymerases have been identified in eukaryotes, and recent investigations have revealed at least sixteen types. An effective approach to study the in vivo roles of each of these is to use selective DNA polymerase inhibitors to distinguish between them. Using a broad natural product screening approach, we have identified many different types of novel DNA polymerase inhibitor which we have exploited to analyze the structure and function of the DNA polymerases. The aim was not only to understand the function of each inhibitor in vitro, but also to develop a drug design strategy for cancer chemotherapy agents. We have found a class of such DNA polymerase inhibitors, the sulfoquinovosylacylglycerols (SQAG), which could represent clinically-promising anti-tumor agents. We discussed about the mechanism of the anti-tumor action, and suggested new concept about cancer chemotherapy.

DNA Polymerase β Catalysis: Are Different Mechanisms Possible?

DNA polymerases are crucial constituents of the complex cellular machinery for replicating and repairing DNA. Discerning mechanistic pathways of DNA polymerase on the atomic level is important for revealing the origin of fidelity discrimination. Mammalian DNA polymerase beta (pol beta), a small (39 kDa) member of the X-family, represents an excellent model system to investigate polymerase mechanisms. Here, we explore several feasible low-energy pathways of the nucleotide transfer reaction of pol beta for correct (according to Watson-Crick hydrogen bonding) G:C basepairing versus the incorrect G:G case within a consistent theoretical framework. We use mixed quantum mechanics/molecular mechanics (QM/MM) techniques in a constrained energy minimization protocol to effectively model not only the reactive core but also the influence of the rest of the enzymatic environment and explicit solvent on the reaction. The postulated pathways involve initial proton abstraction from the terminal DNA primer O3'H group, nucleophilic attack that extends the DNA primer chain, and elimination of pyrophosphate. In particular, we analyze several possible routes for the initial deprotonation step: (i) direct transfer to a phosphate oxygen O(Palpha) of the incoming nucleotide, (ii) direct transfer to an active site Asp group, and (iii) transfer to explicit water molecules. We find that the most probable initial step corresponds to step (iii), involving initial deprotonation to water, which is followed by proton migration to active site Asp residues, and finally to the leaving pyrophosphate group, with an activation energy of about 15 kcal/mol. We argue that initial deprotonation steps (i) and (ii) are less likely as they are at least 7 and 11 kcal/mol, respectively, higher in energy. Overall, the rate-determining step for both the correct and the incorrect nucleotide cases is the initial deprotonation in concert with nucleophilic attack at the phosphate center; however, the activation energy we obtain for the mismatched G:G case is 5 kcal/mol higher than that of the matched G:C complex, due to active site structural distortions. Taken together, our results support other reported mechanisms and help define a framework for interpreting nucleotide specificity differences across polymerase families, in terms of the concept of active site preorganization or the so-called "pre-chemistry avenue".

Inhibitory Effect of Acetogenins on Mammalian DNA Polymerase and Human Cancer Cell Growth

Acetogenins from the Annonaceous plant are a fatty acid-derived natural product. We prepared the chemically synthesized natural acetogenins such as mucocin (compound 1), jimenezin (compound 2), muconin (compound 4), pyranicin (compound 5), and pyragonicin (compound 6). As related studies, 19-epi jimenezin (compound 3), 10-epi pyragonicin (compound 7), and a γ-lactone (compound 8), which is estimated to be a biosynthetic precursor of acetogenins, were also synthesized. Compounds 5 and 6 strongly inhibited, and compounds 1 - 4 and 7 moderately inhibited the activities of mammalian DNA polymerases (pols) such as replicative pol α and repair / recombination-related pol β. On the other hand, compound 8 did not influence the activities of any pols. Compound 5 was the strongest inhibitor of the pols tested, and the IC50 values for pols α and β were 5.3 and 9.6 μM, respectively. These compounds also suppressed human cancer cell (promyelocytic leukemia cell line, HL-60) growth with the same tendency as the inhibition of mammalian pols. Compound 5 was the strongest suppressor of the proliferation of HL-60 cells tested, and the LD50 value was 7.4 μM. The relationship between the three-dimensional molecular structure of acetogenins and these inhibitory activities is discussed.

Mechanism of cell cycle arrest and apoptosis induction by conjugated eicosapentaenoic acid, which is a mammalian DNA polymerase and topoisomerase inhibitor

Conjugated eicosapentaenoic acid (cEPA) selectively inhibited the activities of mammalian DNA polymerases (pols) and human DNA topoisomerases (topos). cEPA inhibited the cell growth of two human leukemia cell lines, NALM-6, which is a p53-wild type, and HL-60, which is a p53-null mutant, with LD50 values of 37.5 and 12.5 microM, respectively. In both cell lines, cEPA arrested in the G1 phase, and increased cyclin E protein levels, indicating that it blocks the primary step of in vivo DNA replication by inhibiting the activity of replicative pols rather than topos. DNA replication-related proteins such as RPA70, ATR and phosphorylated-Chk1/2 were increased by cEPA treatment in the cell lines, suggesting that cEPA led to DNA replication fork stress inhibiting the activities of pols and topos, and the ATR-dependent DNA damage response pathway could respond to the inhibitor of DNA replication. The compound induced cell apoptosis through both p53-dependent and p53-independent pathways in cell lines NALM-6 and HL-60, respectively. These results suggested the therapeutic potential of cEPA as a leading anti-cancer compound that inhibited activities of pols and topos.

A Unified Kinetic Mechanism Applicable to Multiple DNA Polymerases,

After extensive studies spanning over half a century, there is little consensus on the kinetic mechanism of DNA polymerases. Using stopped-flow fluorescence assays for mammalian DNA polymerase beta (Pol beta), we have previously identified a fast fluorescence transition corresponding to conformational closing, and a slow fluorescence transition matching the rate of single-nucleotide incorporation. Here, by varying pH and buffer viscosity, we have decoupled the rate of single-nucleotide incorporation from the rate of the slow fluorescence transition, thus confirming our previous hypothesis that this transition represents a conformational event after chemistry, likely subdomain reopening. Analysis of an R258A mutant indicates that rotation of the Arg258 side chain is not rate-limiting in the overall kinetic pathway of Pol beta, yet is kinetically significant in subdomain reopening. We have extended our kinetic analyses to a high-fidelity polymerase, Klenow fragment (KF), and a low-fidelity polymerase, African swine fever virus DNA polymerase X (Pol X), and showed that they follow the same kinetic mechanism as Pol beta, while differing in relative rates of single-nucleotide incorporation and the putative conformational reopening. Our data suggest that the kinetic mechanism of Pol beta is not an exception among polymerases, and furthermore, its delineated kinetic mechanism lends itself as a platform for comparison of the kinetic properties of different DNA polymerases and their mutants.

A dideoxynucleotide‐sensitive DNA polymerase activity characterized from endoreduplicating cells of mungbean (Vigna radiata L.) during ontogeny of cotyledons

Within this work we describe the purification and biochemical characterization of a ddNTP-sensitive DNA polymerase purified from mungbean (Vigna radiata cv B1, L.) seeds at 18 days after fertilization, when > 70% of the nuclei are reported to be in the endoreduplicated state. The purified enzyme is a single polypeptide of 62 kDa and many of its physicochemical properties are similar to those of mammalian DNA polymerase beta. Similar to the other X-family DNA polymerases, it lacks 3'-5' exonuclease activity and has short gap-filling and strand-displacement activity. The enzyme shows moderately processive DNA synthesis on a single-strand template. The determined N-terminal heptapeptide sequence of the enzyme showed clear homology with helix 1 of the N-terminal single strand DNA-binding domain (residues 32-41) of rat and human DNA polymerase beta. These results represent the first evidence for the identification and characterization of a ddNTP-sensitive DNA polymerase expressed during the endoreduplication cycle that shares biochemical and immunological similarity with mammalian DNA polymerase beta.

A Novel DNA Damage Response

Mammalian DNA polymerase (Pol) delta is essential for DNA replication. It consists of four subunits, p125, p50, p68, and p12. We report the discovery that the p12 subunit is rapidly degraded in cultured human cells by DNA damage or replication stress brought about by treatments with UV, methyl methanesulfonate, hydroxyurea, and aphidicolin. The degradation of p12 is due to an accelerated rate of proteolysis that is inhibited by the proteasome inhibitors, MG132 and lactacystin. UV treatment converts Pol delta in vivo to the three-subunit form lacking p12. This was demonstrated by its isolation using immunoaffinity chromatography. The three-subunit enzyme retains activity on poly(dA)/oligo(dT) templates but is impaired in its ability to extend singly primed M13 templates, clearly indicating that its in vivo functions are likely to be compromised. This transformation of Pol delta by modification of its quaternary structure is reversible in vitro by the addition of the p12 subunit and could represent a novel in vivo mechanism for the modulation of Pol delta function. UV and hydroxyurea-triggered p12 degradation is blocked in ATR(-/-) cells but not in ATM(-/-) cells, thereby demonstrating that p12 degradation is regulated by ATR, the apical kinase that regulates the damage response in S-phase. These findings reveal a novel addition to the cellular repertoire of DNA damage responses that also impacts our understanding of the role of Pol delta in both DNA replication and DNA repair.

Cholesterol hemisuccinate: A selective inhibitor of family X DNA polymerases

Cholesterol hemisuccinate (compound 5), which consists of succinic acid esterified to the β-hydroxyl group of cholesterol, selectively and strongly inhibited the activities of mammalian DNA polymerases (pols) such as pol β, pol λ, and terminal deoxynucleotidyltransferase (TdT), which are family X pols, in vitro, and the IC50 values were 2.9, 6.3, and 6.5μM, respectively. The compound moderately suppressed the activities of other mammalian pols such as pol A (i.e., pol γ), pol B (i.e., pols α, δ, and ε), and pol Y (i.e., pols ι, η, and κ) with 50% inhibition observed at concentrations of 131, 89.2–98.0, and 120–125μM, respectively. The compound had no influence on the activities of plant pols α and β, prokaryotic pols and other DNA metabolic enzymes tested. Since other cholesterol-related compounds such as cholesterol, cholesteryl chloride, cholesteryl bromide, cholesteryl acetate, and cholesteryl-5α, 6α-epoxide (compounds 1–4 and 6, respectively) did not influence the activities of any enzymes tested, the hemisuccinate group of compound 5 could be important for inhibition of the pol X family. Surface plasmon resonance analysis demonstrated that compound 5 bound selectively to the C-terminal 31kDa domain of pol β and pol λ containing a pol β-like region. On the basis of these results, the inhibitory mechanism of compound 5 on the pol X family was discussed.

Telbivudine: a new treatment for chronic hepatitis B.

Three hundred and fifty million people worldwide are estimated to be chronically infected with hepatitis B virus. 15%-40% of these subjects will develop cirrhosis, liver failure or hepatocellular carcinoma during their life. The treatment of chronic hepatitis B has improved dramatically over the last decade merits to the advent of nucleoside/nucleotide analogues and the use of pegylated interferons. Approved drugs for chronic hepatitis B treatment include: standard interferon-alpha 2b, pegylated interferon-alpha 2a, lamivudine, adefovir dipivoxil, and entecavir. Unfortunately, these agents are not effective in all patients and are associated with distinct side effects. Interferons have numerous side effects and nucleoside or nucleotide analogues, which are well tolerated, need to be used for prolonged periods, even indefinitely. However, prolonged treatment with nucleoside or nucleotide analogues is associated with a high rate of resistance. Telbivudine is a novel, orally administered nucleoside analogue for use in the treatment of chronic hepatitis B. In contrast to other nucleoside analogues, Telbivudine has not been associated with inhibition of mammalian DNA polymerase with mitochondrial toxicity. Telbivudine has demonstrated potent activity against hepatitis B with a significantly higher rate of response and superior viral suppression compared with lamivudine, the standard treatment. Telbivudine has been generally well tolerated, with a low adverse effect profile, and at its effective dose, no dose-limiting toxicity has been observed. Telbivudine is one of the most potent antiviral agents for chronic hepatitis B virus and was approved by the FDA in late 2006.

Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase α

In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol α from a fungus (Alternaria tennuis). We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited only mammalian pol α with IC50 value of 0.5μM, and did not influence the activities of other replicative pols such as pols δ and ε, but also showed no effect on pol α activity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. The compound also inhibited the proliferation of human cancer cells with LD50 values of 38.0–44.4μM. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, dehydroaltenusin was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that increased tumor necrosis and decreased mitotic index were apparently detected by the compound in vivo. Therefore, dehydroaltenusin could be of interest as not only a mammalian pol α-specific inhibitor, but also as a candidate drug for anti-cancer treatment.

Kinetic Effect of a Downstream Strand and Its 5′-Terminal Moieties on Single Nucleotide Gap-filling Synthesis Catalyzed by Human DNA Polymerase λ

During short-patch base excision repair, the excision of a 5'-terminal 2-deoxyribose-5-phosphate moiety of the downstream strand by the 5'-2-deoxyribose-5-phosphate lyase activity of either DNA polymerase beta or lambda is believed to occur after each respective enzyme catalyzes gap-filling DNA synthesis. Yet the effects of this 5'-terminal 2-deoxyribose-5-phosphate moiety on the polymerase activities of these two enzymes have never been quantitatively determined. Moreover, x-ray crystal structures of truncated polymerase lambda have revealed that the downstream strand and its 5'-phosphate group of gapped DNA interact intensely with the dRPase domain, but the kinetic effect of these interactions is unclear. Here, we utilized pre-steady state kinetic methods to systematically investigate the effect of a downstream strand and its 5'-moieties on the polymerase activity of the full-length human polymerase lambda. The downstream strand and its 5'-phosphate were both found to increase nucleotide incorporation efficiency (kp/Kd) by 15 and 11-fold, respectively, with the increase procured by the effect on the nucleotide incorporation rate constant kp rather than the ground state nucleotide binding affinity Kd. With 4 single nucleotide-gapped DNA substrates containing a 1,2-dideoxyribose-5-phosphate moiety, a 2-deoxyribose-5-phosphate mimic, we measured the incorporation efficiencies of 16 possible nucleotides. Our results demonstrate that although this 5'-terminal 2-deoxyribose-5-phosphate mimic does not affect the fidelity of polymerase lambda, it moderately decreased the polymerase efficiency by 3.4-fold. Moreover, this decrease in polymerase efficiency is due to a drop of similar magnitude in kp rather than Kd. The implication of the downstream strand and its 5'-moieties on the kinetics of gap-filling synthesis is discussed.

Inhibitory action of C22-fatty acids on DNA polymerases and DNA topoisomerases

We reported previously that unsaturated linear-chain fatty acids of the cis-configuration with a C18-hydrocarbon chain such as linoleic acid (cis-9, 12-octadecadienoic acid, C18:2) could potently inhibit the activity of mammalian DNA polymerases (Biochim Biophys Acta 1308: 256-262, 1996). In this study, we investigated the inhibitory effects of cis-type C22-fatty acids including cis-7,10,13,16,19-docosapentaenoic acid (DPA, C22:5) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA, C22:6) on mammalian DNA polymerases and human DNA topoisomerases. Cis-13,16-docosadienoic acid (C22:2) was the strongest inhibitor of both DNA polymerases and topoisomerases of all C22-fatty acids tested. The inhibitory tendency by the fatty acids on DNA polymerases was the same as that of DNA topoisomerases, and the second strongest inhibitor was cis-13,16,19-docosatrienoic acid (C22:3). The energy-minimized three-dimensional structures of the fatty acids were calculated and it was found that a length of 19-21 Angstrom and width of more than 7 Angstrom in C22-fatty acid structure were important for enzyme inhibition. The three-dimensional structure of the active site of both DNA polymerases and topoisomerases must have a pocket to join C22:2, and this pocket was 19.41 Angstrom long and 9.58 Angstrom wide.

Synthesis of plakevulin A and structure–activity relationships of its related compounds against DNA polymerases

Synthesis of plakevulin A and structure–activity relationships of its related compounds against DNA polymerases is described. We have achieved a total synthesis and revised the structure of plakevulin A. Several analogues including untenone A, manzamenone A, and optically active plakevulin A, were prepared and tested with an enzyme inhibition assay for mammalian DNA polymerases. The effect of the methyl ester moiety, and the substituents at the 1- and 4-positions of plakevulin A on DNA polymerase activities are discussed.

Functional Roles of p12, the Fourth Subunit of Human DNA Polymerase δ

Mammalian DNA polymerase delta (pol delta), a key enzyme of chromosomal DNA replication, consists of four subunits as follows: the catalytic subunit; p125, which is tightly associated with the p50 subunit; p68, a proliferating cell nuclear antigen (PCNA)-binding protein; and a fourth subunit, p12. In this study, the functional roles of the p12 subunit of pol delta were studied. The inter-subunit interactions of the p12 subunit were determined by yeast two-hybrid assays and by pulldown assays. These assays revealed that p12 interacts with p125 as well as p50. This dual interaction of p12 suggests that it may serve to stabilize the p125-p50 interaction. p12 was shown to be a novel PCNA-binding protein. This was confirmed by identification of a PCNA-binding motif at its N terminus by binding assays and by site-directed mutagenesis. The activities and reaction products of recombinant pol delta containing a p12 mutant defective in PCNA binding, as well as purified recombinant pol delta and its subassemblies, were analyzed. Our results indicate that p12 contributes to PCNA-dependent pol delta activity, i.e. the p12-PCNA interaction is functional. Our data indicate that both p12 and p68 are required for optimal pol delta activity. This supports the hypothesis that the interaction between pol delta and PCNA is a divalent one that involves p12 and p68. We propose a model in which pol delta interacts with PCNA via at least two of its subunits, and one in which p12 could play a role in stabilizing the overall pol delta-PCNA complex as well as pol delta itself.

β-Sitosterol-3- O-β- d-glucopyranoside: A eukaryotic DNA polymerase λ inhibitor

β-Sitosterol-3- O-β- d-glucopyranoside (compound 1), a steroidal glycoside isolated from onion ( Allium cepa L.) selectively inhibited the activity of mammalian DNA polymerase λ (pol λ) in vitro. The compound did not influence the activities of replicative DNA polymerases such as α, δ and ɛ, but also showed no effect even on the activity of pol β which is thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. Since parts of compound 1 such as β-sitosterol (compound 2) and d-glucose (compound 3) did not influence the activities of any enzymes tested, the converted structure of compounds 2 and 3 might be important for pol λ inhibition. The inhibitory effect of compound 1 on both intact pol λ (i.e. residues 1–575) and a truncated pol λ lacking the N-terminal BRCA1 C-terminus (BRCT) domain (133–575, del-1 pol λ) was dose-dependent, and 50% inhibition was observed at a concentration of 9.1 and 5.4 μM, respectively. The compound 1-induced inhibition of del-1 pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol λ inhibitory mechanism of compound 1 is discussed.

ArabidopsisTEBICHI, with Helicase and DNA Polymerase Domains, Is Required for Regulated Cell Division and Differentiation in Meristems

In plant meristems, each cell divides and differentiates in a spatially and temporally regulated manner, and continuous organogenesis occurs using cells derived from the meristem. We report the identification of the Arabidopsis thaliana TEBICHI (TEB) gene, which is required for regulated cell division and differentiation in meristems. The teb mutants show morphological defects, such as short roots, serrated leaves, and fasciation, as well as defective patterns of cell division and differentiation in the meristem. The TEB gene encodes a homolog of Drosophila MUS308 and mammalian DNA polymerase theta, which prevent spontaneous or DNA damage-induced production of DNA double strand breaks. As expected from the function of animal homologs, teb mutants show constitutively activated DNA damage responses. Unlike other fasciation mutants with activated DNA damage responses, however, teb mutants do not activate transcriptionally silenced genes. teb shows an accumulation of cells expressing cyclinB1;1:GUS in meristems, suggesting that constitutively activated DNA damage responses in teb lead to a defect in G2/M cell cycle progression. Furthermore, other fasciation mutants, such as fasciata2 and tonsoku/mgoun3/brushy1, also show an accumulation of cells expressing cyclinB1;1:GUS in meristems. These results suggest that cell cycle progression at G2/M is important for the regulation of the pattern of cell division and of differentiation during plant development.

Beatrice A. Singer: In Memoriam (1923–2005)

Dr. Beatrice A. (Bea) Singer died July 20, 2005, after an outstanding career during which she applied her love of high-resolution chemistry to a wide range of topics. She made major contributions to the characterization of tobacco mosaic virus (TMV), RNA and DNA chemistry, alkylation, and other

The In Vitro and In Vivo Inhibitory Effect of Dehydroaltenusin: A Specific Inhibitor of Mammalian DNA Polymerase α

In the screening of the selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol from a fungus (Alternaria tennuis). This compound inhibited only mammalian polα, and did not influence the activities of other replicative pols such as polsαs and Ƹ , but also showed no effect even on polαactivity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence DNA metabolic enzymes tested. The inhibitory effect of dehydroaltenusin on mammalian polαwas dose-dependent with IC50 value of 0.5 ?M. This effect was 10-fold stronger than that of aphidicolin, a well-known potent eukaryotic polαinhibitor. The inhibitory mode of dehydroaltenusin for mammalian polαactivity was competitive with the DNA template-primer and noncompetitive with the dNTP substrate. We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited the cell proliferation of human gastric cancer cell lines by arresting the cells at the S-phase, and preventing the incorporation of thymidine into the cells, indicating that it blocks in vivo DNA replication by inhibiting polα. This compound also induced cell apoptosis. Furthermore, we investigated in vivo anti-tumor effects on nude mice bearing solid tumors of the human cervical cancer cell line HeLa. Dehydroaltenusin was significantly effective in suppressing the growth of solid tumors, therefore, it was of interest as a candidate drug for anti-cancer treatment. These results suggested that dehydroaltenusin is a mammalian polα-specific inhibitor useful in both in vivo and in vitro experiments.