RESUMO
The in vitro relationship between the human p53 DNA binding domain (p53 DBD) and glycolipids was investigated. We isolated the glycolipid fraction from spinach (Spinacia oleracea L.) and found that the fraction inhibited the double-stranded DNA (dsDNA) binding activity of p53 DBD. Since the fraction contained mainly three glycolipids, monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG), and each glycolipid was purified using silica gel column chromatography. Purified SQDG inhibited the activity, however, purified MGDG and DGDG had no influence. In this study, we demonstrated the structure-function relationship between chemically synthetic SQDG and p53 DBD. The major action is probably dependent on the fatty acid effect, although SQDG was a much stronger inhibitor than the fatty acid alone present in SQDG. The inhibitory activity of SQDG was weakened by the R248A mutant of p53 DBD, suggesting that R248 in the dsDNA binding site of p53 must be important for the inhibitory activity of SQDG. SQDG binding to p53 DBD could be reversed with a non-ionic detergent, Nonidet P-40. This is the first study of a glycolipid, SQDG, acting as a dsDNA binding inhibitor of p53, and it could be considered that a SQDG-containing thylakoid membrane in plant chloroplasts might regulate the activity of p53 for cell division, cell cycle checkpoint and tumor suppression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/genética , Glicolipídeos/química , Glicolipídeos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Simulação por Computador , DNA/química , Glicolipídeos/genética , Glicolipídeos/farmacologia , Humanos , Modelos Químicos , Estrutura Molecular , Ligação Proteica , Spinacia oleracea/química , Relação Estrutura-AtividadeRESUMO
The eukaryotic DNA polymerase processivity factor, proliferating cell nuclear antigen, is an essential component in the DNA replication and repair machinery. In Drosophila melanogaster, we cloned a second PCNA cDNA that differs from that encoded by the gene mus209 (for convenience called DmPCNA1 in this article). The second PCNA cDNA (DmPCNA2) encoded a 255 amino acid protein with 51.7% identity to DmPCNA1, and was ubiquitously expressed during Drosophila development. DmPCNA2 was localized in nuclei as a homotrimeric complex and associated with Drosophila DNA polymerase delta and epsilonin vivo. Treatment of cells with methyl methanesulfonate or hydrogen peroxide increased the amount of both DmPCNA2 and DmPCNA1 associating with chromatin, whereas exposure to UV light increased the level of association of only DmPCNA1. Our observations suggest that DmPCNA2 may function as an independent sliding clamp of DmPCNA1 when DNA repair occurs.
Assuntos
Drosophila melanogaster/genética , Antígeno Nuclear de Célula em Proliferação/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Clonagem Molecular , Dano ao DNA/efeitos dos fármacos , DNA Polimerase II/metabolismo , DNA Polimerase III/metabolismo , DNA Complementar/isolamento & purificação , Dimerização , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Modelos Moleculares , Dados de Sequência Molecular , Mutagênicos/farmacologia , Antígeno Nuclear de Célula em Proliferação/isolamento & purificação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
The in vitro relationship between human p53 DNA binding domain (p53 DBD) and FA was investigated. We found that saturated and monounsaturated long-chain FA inhibited the double-stranded DNA (dsDNA) binding activity of p53 DBD. The strongest inhibitors of saturated and unsaturated FA were docosanoic acid (22:0) and cis-12-heneicosenoic acid (21:1n-9), respectively. n-Octadecane, trans-unsaturated FA, and FAME had no influence on the binding activity of p53 DBD, showing that the FA structures such as one or no double bond of cis configuration, hydrocarbon chain of length C20 to C22, and free carboxyl groups are important for the inhibition. The inhibitory effect of the R248A mutant of p53 DBD by saturated FA was as strong as that for wild-type p53 DBD. On the other hand, the inhibition of dsDNA binding activity of the same mutant by the cis-configuration of monounsaturated FA was weaker than that for the wild type. These results suggest that R248 in p53 DBD is important for binding to monounsaturated FA. This is the first report that long-chain FA act as a dsDNA binding inhibitor of p53, and it could be considered that FA in the cell membrane might regulate the activity of p53 for cell division, cell-cycle checkpoint, and tumor suppression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Simulação por Computador , DNA/metabolismo , Humanos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Ligação Proteica , Ressonância de Plasmônio de Superfície/métodos , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
We previously reported the mode of inhibition of DNA polymerase beta (pol. beta) by long chain fatty acids and a bile acid, involving binding analyses to the N-terminal 8-kDa DNA binding domain. Here we describe a site-directed mutational analysis in which the key amino acids (L11, K35, H51, K60, L77, and T79), which are direct interaction sites in the domain, were substituted with K, A, A, A, K, and A, respectively. And their pol. beta interactions with a C24-long chain fatty acid, nervonic acid (NA), and a bile acid, lithocholic acid (LCA), were investigated by gel mobility shift assay and NMR spectroscopy. In the case of K35A, there was complete loss of DNA binding activity while K60A hardly has any activity. In contrast the other mutations had no appreciable effects. Thus, K35 and K60 are key amino acid sites for binding to template DNA. The DNA binding activities of L11K, H51A, and T79A as well as the wild type were inhibited by NA to the same extent. T79A demonstrated a disturbed interaction with LCA. 1H-15N HSQC NMR analysis indicated that despite their many similarities, the wild-type and the mutant proteins displayed some significant chemical shift differences. Not only were the substituted amino acid residues three-dimensionally shifted, but some amino acids which are positioned far distant from the key amino acids showed a shift. These results suggest that the interaction surface was significantly distorted with the result that LCA could not bind to the domain. These findings confirm our previous biochemical and 3D structural proposals concerning inhibition by NA and LCA.
Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , Inibidores Enzimáticos/química , Ácidos Graxos Monoinsaturados/química , Ácido Litocólico/química , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , DNA Polimerase beta/genética , DNA Polimerase beta/isolamento & purificação , Modelos Moleculares , Peso Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RatosRESUMO
It was found that a class of sulfolipids known as sulfo-quinovosyl-acyl-glycerols (SQAGs) from ferns and algae are potent inhibitors of eukaryotic DNA polymerase alpha and beta and effective anti-neoplastic agents. In developing a procedure for the chemical synthesis of sulfolipids, many derivatives and stereoisomers of SQAGs have been obtained including sulfo-quinovosyl-monoacyl-glycerols (SQMGs) and sulfo-quinovosyl-diacyl-glycerols (SQDGs). This review describes studies on the structure-function relationship between synthetic SQAGs and DNA polymerase alpha and beta, and the relationship to cytotoxic activity. The major action was probably dependent on the fatty acid effect, which was reported previously, although each of the SQAGs was a much stronger inhibitor than just the fatty acid present in the SQAGs. The inhibitory effect could be influenced by the chain size of fatty acids in the SQAGs. The sulfonyl group in quinovose was also needed to inhibit the enzymes. Lineweaver-Burk plots of SQAGs indicated that DNA polymerase alpha was non-competitively inhibited, but the SQAGs were effective as antagonists of both the template-primer DNA-binding and the nucleotide substrate-binding of DNA polymerase beta. Based on these results, the molecular actions of SQAGs and drug design strategies for developing new anti-neoplastic agents were discussed.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Glicerol/análogos & derivados , Monossacarídeos/farmacologia , Inibidores da Síntese de Ácido Nucleico , Animais , Antineoplásicos/química , Células Cultivadas , Inibidores Enzimáticos/química , Glicerol/química , Glicerol/farmacologia , Glicolipídeos , Modelos Moleculares , Estrutura Molecular , Monossacarídeos/químicaRESUMO
We have previously reported that sulfoquinovosylmonoacylglycerol (SQMG) is a potent inhibitor of mammalian DNA polymerases. DNA polymerase beta (pol beta) is one of the most important enzymes protecting the cell against DNA damage by base excision repair. In this study, we characterized the inhibitory action of SQMG against rat pol beta. SQMG competed with both the substrate and the template-primer for binding to pol beta. A gel mobility shift assay and a polymerase activity assay showed that SQMG competed with DNA for a binding site on the N-terminal 8-kDa domain of pol beta, subsequently inhibiting its catalytic activity. Fragments of SQMG such as sulfoquinovosylglycerol (SQG) and fatty acid (myristoleic acid, MA) weakly inhibited pol beta activity and the inhibitory effect of a mixture of SQG and MA was stronger than that of SQG or MA. To characterize this inhibition more precisely, we attempted to identify the interaction interface between SQMG and the 8-kDa domain by NMR chemical shift mapping. Firstly, we determined the binding site on a fragment of SQMG, the SQG moiety. We observed chemical shift changes primarily at two sites, the residues comprising the C-terminus of helix-1 and the N-terminus of helix-2, and residues in helix-4. Finally, based on our present results and our previously reported study of the interaction interface of fatty acids, we constructed two three-dimensional models of a complex between the 8-kDa domain and SQMG and evaluated them by the mutational analysis. The models show a SQMG interaction interface that is consistent with the data.
Assuntos
DNA Polimerase beta/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glicolipídeos/farmacologia , Animais , Sítios de Ligação , Ligação Competitiva , DNA/biossíntese , DNA Polimerase beta/química , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Glicolipídeos/química , Glicolipídeos/metabolismo , Técnicas In Vitro , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Studies of mammalian terminal deoxyribonucleotidyltransferase (TdT) are facilitated by use of inhibitors that selectively knock down the activity of the enzyme. We have screened for selective inhibitors of TdT and identified a natural compound with this property in the Japanese vegetable, Arctium lappa. The compound has little effect on the activities of mammalian DNA polymerases, such as alpha, beta, delta or lambda polymerase, and prokaryotic DNA polymerases, such as Taq DNA polymerase, T4 DNA polymerase and Klenow fragment. H1- and C13-NMR spectroscopic analyses showed the compound to be baicalin, a compound previously reported as an anti-inflammatory or antipyretic agent. The IC50 value of baicalin to TdT was 18.6 microM. We also found that genistin, a baicalin derivative known to be antimutagenic, more selectively inhibited TdT activity than baicalin, although its IC50 value was weaker (28.7 microM). Genistin and baicalin also inhibited the activity of truncated TdT (the so-called pol beta core domain) in which the BRCT motif was deleted in its N-terminal region. In kinetic analyses, inhibition by either genistin or baicalin was competitive with the primer and non-competitive with the dNTP substrate. The compounds may, therefore, bind directly to the primer-binding site of TdT and simultaneously disturb dNTP substrate incorporation into the primer. Genistin and baicalin should prove to be useful agents for studying TdT.
Assuntos
DNA Nucleotidilexotransferase/antagonistas & inibidores , Flavonoides/farmacologia , Isoflavonas/farmacologia , Teprotida/farmacologia , Animais , DNA Polimerase I/antagonistas & inibidores , DNA Polimerase III/antagonistas & inibidores , DNA Polimerase beta/antagonistas & inibidores , Primers do DNA , Flavanonas/farmacologia , Humanos , Cinética , Poli C/farmacologia , Ratos , Soroalbumina Bovina/farmacologia , Taq Polimerase/antagonistas & inibidoresAssuntos
Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Proteínas de Grupo de Alta Mobilidade/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Fatores de Elongação da Transcrição/química , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Proteínas de Grupo de Alta Mobilidade/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Subunidades Proteicas/genética , Fatores de Elongação da Transcrição/genéticaRESUMO
Previously, we described a novel DNA polymerase, designated as OsPolI-like, from rice. The OsPolI-like showed a high degree of sequence homology with the DNA polymerase I of cyanobacteria and was localized in the plastid. Here, we describe two PolI-like polymerases, designated as AtPolI-like A and AtPolI-like B, from Arabidopsis thaliana. In situ hybridization analysis demonstrated expression of both mRNAs in proliferating tissues such as the shoot apical meristem. Analysis of the localizations of GFP fusion proteins showed that AtPolI-like A and AtPolI-like B were localized to plastids. AtPolI-like B expression could be induced by exposure to the mutagen H(2)O(2). These results suggested that AtPolI-like B has a role in the repair of oxidation-induced DNA damage. Our data indicate that higher plants possess two plastid DNA polymerases that are not found in animals and yeasts.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Plastídeos/genética , Plastídeos/metabolismo , Sequência de Aminoácidos , DNA Polimerase I/química , Dados de Sequência Molecular , Estruturas Vegetais/genética , Estruturas Vegetais/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Translocated in liposarcoma (TLS) is an important protein component of the heterogeneous nuclear ribonucleoprotein complex involved in the splicing of pre-mRNA and the export of fully processed mRNA to the cytoplasm. We examined the domain organization of human TLS by a combined approach using limited proteolysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, circular dichroism, inductively coupled plasma atomic emission spectroscopy, and NMR spectroscopy. We found that the RNA recognition motif (RRM) and zinc finger-like domains exclusively form protease-resistant core structures within the isolated TLS protein fragments, while the remaining regions, including the Arg-Gly-Gly repeats, appear to be completely unstructured. Thus, TLS contains the unstructured N-terminal half followed by the RRM and zinc finger-like domains, which are connected to each other by a flexible linker. We also carried out NMR analyses to obtain more detailed insights into the individual RRM and zinc finger-like domains. The 113Cd NMR analysis of the zinc finger-like domain verified that zinc is coordinated with four cysteines in the C4 type scheme. We also investigated the interaction of each domain with an oligo-RNA containing the GGUG sequence, which appears to be critical for the TLS function in splicing. The backbone amide NMR chemical shift perturbation analyses indicated that the zinc finger domain binds GGUG-containing RNA with a dissociation constant of about 1.0 x 10(-5) m, whereas the RRM domain showed no observable interaction with this RNA. This surprising result implies that the zinc finger domain plays a more predominant role in RNA recognition than the RRM domain.
Assuntos
Proteína FUS de Ligação a RNA/química , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Quimotripsina/farmacologia , Dicroísmo Circular , Cisteína/química , Citoplasma/metabolismo , DNA Complementar/metabolismo , Glutationa Transferase/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , RNA/química , Proteína FUS de Ligação a RNA/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Atômica , Dedos de ZincoRESUMO
We reported previously that lithocholic acid (LCA, 3-alpha-hydroxy-5-beta-cholan-24-oic acid), one of the major compounds in the secondary bile acids, selectively inhibited the activity of mammalian DNA polymerase beta (pol beta) [Mizushina, Y., Ohkubo, T., Sugawara, F., and Sakaguchi, K. (2000) Biochemistry 39, 12606-12613]. The purpose of this study was to investigate the molecular structural relationship of LCA and its 10 chemically synthesized derivatives. The inhibitory activities of pol beta by some derivative compounds were stronger than that by LCA, and these compounds bound tightly to the 8-kDa domain fragment but not to the 31-kDa domain fragment of pol beta. Biacore analysis demonstrated that the 8-kDa domain bound selectively to compound 9 (3-alpha-O-lauroyl-5-beta-cholan-24-oic acid), which was the strongest pol beta inhibitor tested, as a 1:1 complex with a dissociation constant (K(d)) of 1.73 nM. From computer modeling analysis (i.e., molecular dynamics analysis), the 8-kDa domain had two inhibitor binding areas. Three amino acid residues (Lys60, Leu77, and Thr79) of the 8-kDa domain bound to LCA and compound 2 (3-alpha-methoxy-5-beta-cholan-24-oic acid), and four amino acid residues (Leu11, Lys35, His51, and Thr79) of the 8-kDa domain bound to compound 9. From these results, the structure-function relationship among pol beta and its selective inhibitors was discussed.
Assuntos
DNA Polimerase beta/química , Ácido Litocólico/química , Animais , Sítios de Ligação , Simulação por Computador , DNA Polimerase beta/antagonistas & inibidores , DNA Polimerase beta/metabolismo , Inibidores Enzimáticos/química , Ácido Litocólico/metabolismo , Ácido Litocólico/farmacologia , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Estrutura Terciária de Proteína , RatosRESUMO
In the basidiomycete Coprinus cinereus (C. cinereus), which shows a highly synchronous meiotic cell cycle, the meiotic prophase I cells demonstrate flap endonuclease-1 activity. To investigate its role during meiosis, we isolated a C. cinereus cDNA homolog of flap endonuclease-1 (CcFEN-1), 1377bp in length with the open reading frame (ORF) encoding a predicted molecular mass of 51 kDa. At amino-acid residues Glu276-Pro345, a specific inserted sequence composed of 70 amino acids rich in polar forms was found to exist, without sequence identity to other eukaryotic FEN-1 or the polar amino acid rich sequences found in C. cinereus PCNA and C. cinereus DNA ligase IV, although the lengths and percentages of polar amino acids were similar. Northern hybridization analysis indicated CcFEN-1 to be expressed not only in the pre-meiotic S phase but also in meiotic prophase I. The roles of CcFEN-1 during meiosis are discussed.
Assuntos
Coprinus/enzimologia , Coprinus/genética , Endonucleases Flap/genética , Regulação Fúngica da Expressão Gênica , Meiose/fisiologia , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Complementar/química , DNA Complementar/isolamento & purificação , DNA Fúngico/química , DNA Fúngico/isolamento & purificação , Endonucleases Flap/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Filogenia , Antígeno Nuclear de Célula em Proliferação/genética , Prófase/genética , Prófase/fisiologia , RNA Fúngico/análise , RNA Mensageiro/análise , Fase S/genética , Fase S/fisiologia , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. Previously, we isolated and characterized a complementary DNA (cDNA) from rice (Oryza sativa) encoding a protein which shows homology with the eukaryotic flap endonuclease-1 (FEN-1). In this report, we found that rice (O. sativa L. cv. Nipponbare) possessed two FEN-1 homologues designated as OsFEN-1a and OsFEN-1b. The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and 3, respectively. Both genes contained 17 exons and 16 introns. Alignment of OsFEN-1a protein with OsFEN-1b protein showed a high degree of sequence similarity, particularly around the N and I domains. Northern hybridization and in situ hybridization analysis demonstrated preferential expression of OsFEN-1a and OsFEN-1b in proliferating tissues such as the shoot apical meristem or young leaves. The levels of OsFEN-1a and OsFEN-1b expression were significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at high level. These results suggested that OsFEN-1a and OsFEN-1b are required for cell proliferation. Functional complementation assay suggested that OsFEN-1a cDNA had the ability to complement Saccharomyces cerevisiae rad27 null mutant. On the other hand, OsFEN-1b cDNA could not complement the rad27 mutant. The roles of OsFEN-1a and OsFEN-1b in plant DNA replication and repair are discussed.
Assuntos
Endonucleases Flap/genética , Oryza/genética , Sequência de Aminoácidos , Divisão Celular/genética , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Clonagem Molecular , Éxons , Endonucleases Flap/química , Endonucleases Flap/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Teste de Complementação Genética , Hibridização In Situ , Íntrons , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutação , Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
DNA ligase IV is thought to be involved in DNA double-strand break repair and DNA non-homologous end-joining pathways, but these mechanisms are still unclear. To investigate the roles of DNA ligase IV from a biologically functional viewpoint, the authors studied its relationship to meiosis in a basidiomycete, Coprinus cinereus, which shows a highly synchronous meiotic cell cycle. The C. cinereus cDNA homologue of DNA ligase IV (CcLIG4) was successfully cloned. The 3.2 kb clone including the ORF encoded a predicted product of 1025 amino acid residues with a molecular mass of 117 kDa. A specific inserted sequence composed of 95 amino acids rich in aspartic acid and glutamic acid could be detected between tandem BRCT domains. The inserted sequence had no sequence identity with other eukaryotic counterparts of DNA ligase IV or with another aspartic acid and glutamic acid rich sequence inserted in C. cinereus proliferating cell nuclear antigen (CcPCNA), although the length and the percentages of aspartic and glutamic acids were similar. In addition, the recombinant CcLIG4 protein not only showed ATP-dependent ligase activity, but also used (dT)(16)/poly(dA) and (dT)(16)/poly(rA) as substrates, and had double-strand ligation activity, like human DNA ligase IV. Northern hybridization analysis and in situ hybridization indicated that CcLIG4 was expressed not only at the pre-meiotic S phase but also at meiotic prophase I. Intense signals were observed in leptotene and zygotene. Based on these observations, the possible role(s) of C. cinereus DNA ligase IV during meiosis are discussed.
Assuntos
Coprinus/enzimologia , DNA Ligases/metabolismo , Alquilantes/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Coprinus/citologia , Coprinus/genética , DNA Ligase Dependente de ATP , DNA Ligases/química , DNA Ligases/genética , Reparo do DNA , DNA Complementar/genética , DNA Fúngico/genética , Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Humanos , Meiose , Metanossulfonato de Metila/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We found that some triterpene compounds could not only selectively inhibit the activities of mammalian DNA polymerase alpha (pol alpha) and beta (pol beta), but could also potently inhibit DNA topoisomerase II (topo II) [Biochem. J. 350 (2000) 757]. Here, we report that natural triterpenes produced by callus from an ancient Chinese medicinal plant were also inhibitors of the enzymes, and some were more selective than others. The natural triterpenes with a carboxyl group equally inhibited the activities of pol alpha, pol beta, and topo II, while the olide-type triterpenes with a ketone group suppressed the activities of pol beta and topo II, but not pol alpha. The other triterpenes from the callus hardly influenced these enzyme activities. As also described previously [J. Biochem. 130 (2001) 657], pol beta and topo II have a three-dimensionally similar triterpene-binding region, which is a pocket in which specific compounds can insert. The newly found triterpene inhibitors might structure-dependently insert into the pocket, and the pocket structure of each enzyme might, three-dimensionally but slightly, differ among them. The triterpene frames could be used for screening new inhibitors of the enzymes, and computer-simulated drug design using the frame and pocket structure may in theory be a possible approach to develop new inhibitors.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Plantas Medicinais/química , Triterpenos/farmacologia , DNA Polimerase I/metabolismo , DNA Polimerase beta/metabolismo , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Plantas Medicinais/crescimento & desenvolvimento , Relação Estrutura-Atividade , Triterpenos/químicaRESUMO
Hippospongic acid A (HA-A) is a novel natural triterpene metabolite that exhibits inhibitory activity against the gastrulation of starfish embryos isolated from a marine sponge, Hippospongia sp. We succeeded in chemically synthesizing the natural enantiomer and the racemate HA-A. In this study, we examined its action mode in vitro. HA-A was a rare compound that could selectively but uniformly inhibit the activities of all the vertebrate DNA polymerases tested such as alpha, beta, delta, epsilon, eta, kappa, and lambda, in the IC(50) range of 5.9-17.6 microM, and interestingly also those of human DNA topoisomerases I and II (IC(50) = 15-25 microM). HA-A exhibited no inhibitory effect on DNA polymerases from insects, plants and prokaryotes, or on many other DNA metabolic enzymes. HA-A was an inhibitor specific to DNA polymerases and DNA topoisomerases from vertebrates, but not selective as to a subclass species among the enzymes. Since DNA polymerase beta is the smallest, we used it to analyze the biochemical relationship with HA-A. Biochemical, BIAcore and computer modeling analyses demonstrated that HA-A bound selectively to the N-terminal 8 kDa DNA template-binding domain of DNA polymerase beta, and HA-A inhibited the ssDNA binding activity. HA-A could prevent the growth of NUGC-3 cancer cells at both the G1 and G2/M phases, and induce apoptosis in the cells. The LD(50) value was 9.5 microM, i.e. in the same range as for the enzyme inhibition. Therefore, we concluded that one molecular basis of the gastrulation of starfish embryos is a process that requires DNA polymerases and DNA topoisomerases, and subsequently the gastrulation was inhibited by HA-A. We also discussed the in vivo role of HA-A.
Assuntos
Ácidos Carboxílicos/metabolismo , Gástrula/metabolismo , Estrelas-do-Mar/embriologia , Triterpenos/metabolismo , Animais , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , DNA Topoisomerases/genética , DNA Topoisomerases/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Gástrula/efeitos dos fármacos , Humanos , Técnicas In Vitro , Modelos Moleculares , Inibidores da Síntese de Ácido Nucleico , Poríferos/metabolismo , Estrelas-do-Mar/metabolismo , Estereoisomerismo , Inibidores da Topoisomerase , Triterpenos/química , Triterpenos/farmacologiaRESUMO
The pyrrolidine alkaloids mimicking the structures of pentose with nitrogen in the ring are known to be inhibitors of glycosidases. We report here that a compound belonging to this category is an inhibitor of eukaryotic DNA polymerases. Among the eight naturally occurring pyrrolidine alkaloids we tested, only one compound, 1,4-dideoxy-1,4-imino-D-ribitol (DRB), which was purified from the mulberry tree (Morus alba), strongly inhibited the activities of eukaryotic DNA polymerases with IC50 values of 21-35 microM, and had almost no effect on the activities of prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase, and bovine deoxyribonuclease I. Kinetic studies showed that inhibition of both DNA polymerases alpha and beta by DRB was competitive with respect to dNTP substrate. Whereas DNA polymerase alpha inhibition was noncompetitive with the template-primer, the inhibition of DNA polymerase beta was found to be competitive with the template-primer. The K(i) values of DNA polymerases alpha and beta for the template-primer were smaller than those for dNTP substrate. Therefore, the affinity of DRB was suggested to be higher at the template-primer binding site than at the dNTP substrate-binding site, although DRB is an analogue of deoxyribose consisting of dNTP. Computational analyses of the eight pyrrolidine alkaloids revealed a remarkable difference in the distribution of positive and negative electrostatic charges on the surface of molecules. The relationship between the structure of DRB and the inhibition of eukaryotic DNA polymerases is discussed.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Álcoois Açúcares/farmacologia , Alcaloides/química , Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Animais , Arabinose , DNA Polimerase I/metabolismo , DNA Polimerase beta/metabolismo , Inibidores Enzimáticos/química , Células Eucarióticas/enzimologia , Humanos , Imino Furanoses , Cinética , Modelos Moleculares , Morus/química , Pirrolidinas/química , Pirrolidinas/isolamento & purificação , Pirrolidinas/farmacologia , Eletricidade Estática , Álcoois Açúcares/químicaRESUMO
Petasiphenol, a bio-antimutagen isolated from a Japanese vegetable, Petasites japonicus, selectively inhibits the activities 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 pol beta activity, the three-dimensional structure of which is thought to be highly similar to pol lambda. The inhibitory effect of petasiphenol on intact pol lambda including the BRCA1 C-terminus (BRCT) domain was dose-dependent, and 50% inhibition was observed at a concentration of 7.8 microM. The petasiphenol-induced inhibition of the pol lambda activity was noncompetitive with respect to both the DNA template-primer and the dNTP substrate. Petasiphenol did not only inhibit the activity of the truncated pol lambda including the pol beta-like core, in which the BRCT motif was deleted in its N-terminal region. BIAcore analysis demonstrated that petasiphenol bound selectively to the N-terminal domain of pol lambda but did not bind to the C-terminal region. On the basis of these results, the pol lambda inhibitory mechanism of petasiphenol is discussed.
Assuntos
Ácidos Cafeicos/química , Ácidos Cafeicos/farmacologia , DNA Polimerase beta/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Sequência de Aminoácidos , Animais , Antimutagênicos/química , Antimutagênicos/isolamento & purificação , Antimutagênicos/farmacologia , Proteína BRCA1/química , Sítios de Ligação , Ácidos Cafeicos/isolamento & purificação , Bovinos , Simulação por Computador , DNA Polimerase beta/química , Inibidores Enzimáticos/isolamento & purificação , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Petasites/química , Estrutura Terciária de Proteína , Ratos , Homologia de Sequência de AminoácidosRESUMO
Previously, we reported the three-dimensional molecular interactions of nervonic acid (NA) with mammalian DNA polymerase beta (pol beta) [Mizushina et al. (1998) J. Biol. Chem. 274, 25599-25607]. By three-dimensional structural model analysis and comparison with the spatial positioning of specific amino acids binding to NA on pol beta (Leu11, Lys35, His51, and Thr79), we obtained supplementary information that allowed us to build a structural model of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). In HIV-1 RT, Leu100, Lys65, His235, and Thr386 corresponded to these four amino acid residues. These results suggested that the NA binding domains of pol beta and HIV-1 RT are three-dimensionally very similar. The effects of NA on HIV-1 RT are thought to be same as those on pol beta in binding to the rhombus of the four amino acid residues. NA dose-dependently inhibited the HIV-1 RT activity. For binding to pol beta, the kinetics were competitive when the rhombus was present on the DNA binding site. However, as the rhombus in HIV-1 RT was not present in the DNA binding site, the three-dimensional structure of the DNA binding site must be distorted, and subsequently the enzyme is inhibited non-competitively.
Assuntos
DNA Polimerase beta/antagonistas & inibidores , Ácidos Graxos Monoinsaturados/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , DNA Polimerase beta/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Evolução Molecular , Ácidos Graxos Monoinsaturados/metabolismo , Transcriptase Reversa do HIV/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , RatosRESUMO
In the present study, we have found that mono-unsaturated linear-chain fatty acids in the cis configuration with C(18) hydrocarbon chains (i.e. oleic acid) strongly inhibited the activity of human telomerase in a cell-free enzymic assay, with an IC(50) value of 8.6 microM. Interestingly, fatty acids with hydrocarbon chain lengths below 16 or above 20 carbons substantially decreased the potency of inhibition of telomerase. Moreover, the cis-mono-unsaturated C(18) linear-chain fatty acid oleic acid was the strongest inhibitor of all the fatty acids tested. A kinetic study revealed that oleic acid competitively inhibited the activity of telomerase ( K (i)=3.06 microM) with respect to the telomerase substrate primer. The energy-minimized three-dimensional structure of the linear-chain fatty acid was calculated and modelled. A molecule width of 11.53-14.26 A (where 1 A=0.1 nm) in the C(16) to C(20) fatty acid structure was suggested to be important for telomerase inhibition. The three-dimensional structure of the telomerase active site (i.e. the substrate primer-binding site) appears to have a pocket that could bind oleic acid, with the pocket being 8.50 A long and 12.80 A wide.