Masaharu Takemura

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

Beta-sitosterol-3-O-beta-D-glucopyranoside (compound 1), a steroidal glycoside isolated from onion (Allium cepa L.) selectively inhibited the activity of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta and epsilon, but also showed no effect even on the activity of pol beta which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of compound 1 such as beta-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 lambda inhibition. The inhibitory effect of compound 1 on both intact pol lambda (i.e. residues 1-575) and a truncated pol lambda lacking the N-terminal BRCA1 C-terminus (BRCT) domain (133-575, del-1 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 9.1 and 5.4 microM, respectively. The compound 1-induced inhibition of del-1 pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol lambda inhibitory mechanism of compound 1 is discussed.

Evolution of Eukaryotic DNA Polymerases via Interaction Between Cells and Large DNA Viruses

B-family DNA-directed DNA polymerases are DNA replication enzymes found in Eukaryota, Archaea, large DNA viruses, and in some, but not all, bacteria. Several polymerase domains are conserved among the B-family DNA polymerases from these organisms, suggesting that the B-family DNA polymerases evolved from a common ancestor. Eukaryotes retain at least three replicative B-family DNA polymerases, DNA polymerase α, δ, and ε, and one translesion B-family DNA polymerase, DNA polymerase ζ. Here, we present molecular evolutionary evidence that suggests DNA polymerase genes evolved through horizontal gene transfer between the viral and archaeal–eukaryotic lineages. Molecular phylogenetic analyses of the B-family DNA polymerases from nucleo-cytoplasmic large DNA viruses (NCLDVs), eukaryotes, and archaea suggest that different NCLDV lineages such as Poxviridae and Mimiviridae were involved in the evolution of different DNA polymerases (pol-α-, δ-, ε-, and ζ-like genes) in archaeal–eukaryotic cell lineages, putatively through horizontal gene transfer. These results support existing theories that link the evolution of NCLDVs and the origin of the eukaryotic nucleus.

Structure and activity relationship of monogalactosyl diacylglycerols, which selectively inhibited in vitro mammalian replicative DNA polymerase activity and human cancer cell growth.

The glycoglycerolipid, monogalactosyl diacylglycerol (MGDG), containing two alpha-linolenic acids (C18:3), was isolated from bitter melon as a potent and selective inhibitor of mammalian DNA polymerase (pol) species such as pols alpha, gamma, delta, and epsilon with IC(50) values of 17.6-37.2muM. This MGDG also suppressed the growth of six human cancer cell lines, although normal human cell lines were not affected. This compound (i.e., MGDG-C18:3-C18:3) was a stronger inhibitor than both MGDG-C18:1-C18:0 and MGDG-C18:0-C18:0. In this study, we discussed the structure-function relationship in the selective inhibition of mammalian replicative pols and human cancer cell proliferation by MGDGs.

Inhibitory effect of coenzyme Q1 on eukaryotic DNA polymerase γ and DNA topoisomerase II activities on the growth of a human cancer cell line

Coenzyme Q (CoQ) is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ having shorter isoprenoid chains, especially CoQ1 and CoQ2, selectively inhibited the in vitro activity of eukaryotic DNA polymerase (pol) γ, which is a mitochondrial pol. These compounds did not influence the activities of nuclear DNA replicative pols such as α, δ and ɛ, and nuclear DNA repair‐related pols such as β, η, ι, κ and λ. CoQ also inhibited DNA topoisomerase II (topo II) activity, although the enzymatic characteristics, including modes of action, amino acid sequences and three‐dimensional structures, were markedly different from those of pol γ. These compounds did not inhibit the activities of procaryotic pols such as Escherichia coli pol I, and other DNA metabolic enzymes such as human immunodeficiency virus reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. CoQ1, which has the shortest isoprenoid chains, had the strongest inhibitory effect on pol γ and topo II activities among CoQ1–CoQ10, with 50% inhibitory concentration (IC50) values of 12.2 and 15.5 µM, respectively. CoQ1 could prevent the growth of human promyelocytic leukemia cells, HL‐60, and the 50% lethal dose (LD50) value was 14.0 µM. The cells were halted at S phase and G1 phase in the cell cycle, and suppressed mitochondrial proliferation. From these results, the relationship between the inhibition of pol γ/topo II and cancer cell growth by CoQ is discussed. (Cancer Sci 2006; 97: 716–723)

Inhibitory action of conjugated C18-fatty acids on DNA polymerases and DNA topoisomerases

We reported previously that unsaturated linear-chain FA of the cis-configuration with a C18-hydrocarbon chain such as linoleic acid (18∶2Δ9c, 12c) could potently inhibit the activities of mammalian DNA polymerases and DNA topoisomerases, but their saturated forms could not. There are chemically two classes of unsaturated FA, normal and conjugated, but only the conjugated forms show potent antitumor activity. In this report, we study the inhibitory effects of chemically synthesized conjugated C18-FA on mammalian DNA polymerases and DNA topoisomerases as compared with normal unsaturated FA. The conjugated α-eleostearic acid (18∶3Δ9c, 11t, 13t) was the strongest of all the FA tested. For the inhibition, the conjugated form is crucially important. The energy-minimized 3-D structures of the FA were calculated, and both a length of less than 20 Å and a width of 8.13–9.24 Å in the C18-FA structure were found to be important for enzyme inhibition. The 3-D structure of the active site of both DNA polymerases and topoisomerases must have had a pocket to join α-eleostearic acid, and this pocket was 12.03 Å long and 9.24 Å wide.

Morphological and Taxonomic Properties of Tokyovirus, the First Marseilleviridae Member Isolated from Japan

Members of the Marseilleviridae family are large DNA viruses with icosahedral particle structures that infect Acanthamoeba cells. The first Marseillevirus to be discovered was isolated in 2009. Since then, several other members of the Marseilleviridae family have been reported, including Lausannevirus, Senegalvirus, Cannes 8 virus, Insectomime virus, Tunisvirus, Melbournevirus, Port-Miou virus, and Brazilian Marseillevirus, which have been isolated from Europe, Africa, Australia, and South America. The morphological and genomic properties of a new Marseilleviridae family member, Tokyovirus, discovered in a water/soil sample from a Japanese river in Tokyo, were described in the present study. Tokyovirus possesses icosahedral particles of up to 200 nm in diameter, as revealed by a transmission electron microscopy (TEM) analysis, which form a giant virion factory in Acanthamoeba cells. A preliminary genome analysis predicted 487 coding sequences. A dot plot analysis and phylogenetic analysis using family B DNA polymerase, proliferating cell nuclear antigen (PCNA), and DNA-directed RNA polymerase alpha subunit genes revealed that Tokyovirus shares similarities with Marseillevirus, Melbournevirus, and Cannes 8 virus (Marseilleviridae subclade A), but not with Lausannevirus and Port-Miou virus (subclade B), Tunisvirus and Insectomime virus (subclade C), or Brazilian Marseillevirus (subclade D), suggesting that Tokyovirus has evolved separately from the previously described Marseilleviridae members.

Chemical properties of fatty acid derivatives as inhibitors of DNA polymerases

In this study, the chemical properties of organic acids as DNA polymerase inhibitors were examined. In total, we assayed the inhibitory activities of 23 compounds. We found that the DNA synthesis activity of DNA polymerase was usually reduced to less than 50% in the presence of 100 microM monoprotic acids, which have a Clog P value greater than 7.0 and a pK(a) value less than 5.4. With a minor modification these chemical properties applied to several organic fatty acids previously reported as DNA polymerase inhibitors. Moreover, we also examined the inhibitory activities of perfluorooctadecanoic acid (PFOdA) and perfluorooctanesulfonic acid (PFOS) against DNA polymerase beta in detail. These compounds inhibited the polymerase activity of pol beta competitively with template-primer DNA, and non-competitively with dNTPs. In addition, the 8 kDa domain-defective pol beta was also sensitive to these compounds. Our results suggest that the inhibitory mode of action of PFOdA and PFOS is different from that mediated by the classic fatty acid inhibitors against DNA polymerase beta.

Draft Genome Sequence of Tokyovirus, a Member of the Family Marseilleviridae Isolated from the Arakawa River of Tokyo, Japan

ABSTRACT Members of the Marseilleviridae family are large DNA viruses with icosahedral particles that infect Acanthamoeba cells. This report presents a new Marseilleviridae family member discovered in a water/soil sample from a river in Tokyo, named Tokyovirus, with genome size of 370 to 380 kb.

Improved “Origami Bird” Protocol Enhances Japanese Students’ Understanding of Evolution by Natural Selection: a Novel Approach Linking DNA Alteration to Phenotype Change

Many studies of evolution education have attempted to develop teaching materials on natural selection because of its importance in the evolutionary process (Lauer 2000; Heim 2002; Catley 2006; Kalinowski et al. 2006; Christensen-Dalsgaard and Kanneworff 2008; Frey et al. 2010). However, to our knowledge, no teaching material effectively connects DNA sequencing to the process of natural selection, although current evolutionary biology is closely linked to molecular biology. Current educational content must be modernized to advance evolution education (Hills 2007). Japanese people easily accept evolution as a fact when compared to people in other countries, especially the U.S. (Sakura 1998; Miller et al. 2006). However, there are some problems in evolution education in Japan, such as using textbooks with outdated scientific theories and a lack of teaching material. These textbooks often cite outdated theories (Shimada 1997, 2004; Nakai 2004), offer a shortage of examples of microevolution (Yamanoi 2008), and include an inaccurate definition of mutation (Yamanoi and Sakura 2010). Fewer teaching materials for evolution have been developed in Japan than in the U.S. and the UK and are hardly introduced in Japanese biology textbooks, particularly for evolutionary mechanisms such as natural selection (Sato and Ohshika 2005). Misconceptions about evolution among high school students are also reported in certain studies. Even after studying evolution, many students fail to understand modern evolutionary concepts, such as linking genes to phenotype, and they mistakenly hold Lamarckism and orthogenesis to be the evolutionary mechanisms (Yamanoi 2008; Fukui 2000; Yamanoi 2010). These misconceptions may be derived from inadequate understanding of the random process in the evolutionary mechanism (Garvin-Doxas and Klymkowsky 2008). We believed that if the students regard mutation as random, not a purposely designed process, then they will disregard Lamarckism and orthogenesis since both ideas assume evolution as a progressive, teleologically designed process. Biology education based on the new Japanese national curriculum framework, the course of study (CS), and its guidelines for secondary school will begin in high schools in 2013. One of the main educational goals in the CS is to relate evolutionary biology to molecular biology (Ministry T. Yamanoi Hakuoh Ashikaga Senior High School, Tadaki-cho1067, Ashikaga, Tochigi 329-4214, Japan

Hyper-Phosphorylated Retinoblastoma Protein Suppresses Telomere Elongation

Immortalized cell lines maintain telomeres by the expression of telomerase or by a mechanism designated alternative lengthening of telomeres (ALT). Although DNA polymerase α (pol-α) is reported to be required for telomere maintenance, the critical role of pol-α in telomere maintenance has not been firmly determined. We examined the role of retinoblastoma protein (pRb) and pol-α in the regulation of telomere length, using telomere-fiber FISH. Telomere length varied dependent on the intracellular abundance of pol-α or pRb in HeLa cells. A proportion of hyper-phosphorylated pRb (ppRb) molecules localized to sites of telomeric DNA replication in HeLa cells. Pol-α might thus contribute to telomere maintenance, and might be regulated by ppRb.