RESUMO
This work describes the synthesis of hybrid tetrahydro-1,5-naphthyridine and 1,5-naphthyridine derivatives fused with heterocycles such as chromenes and chromen-2-ones or coumarins, which were synthesized in good to high general yields. The synthetic route involves an intramolecular [4 + 2]-cycloaddition reaction of functionalized aldimines obtained by the condensation of 3-aminopyridine and aldehydes containing a double or triple carbon-carbon bond in orto position and allows the selective generation of three stereogenic centers in a short, efficient and reliable synthesis. The subsequent dehydrogenation of the fused tetrahydrochromeno[4,3-b][1,5]naphthyridines and/or tetrahydrochromeno[4,3-b][1,5]naphthyridin-6-ones leads to the formation of the corresponding tetracyclic chromeno[4,3-b][1,5]naphthyridine derivatives and/or chromeno[4,3-b][1,5]naphthyridin-6-ones in quantitative yields. Some of the prepared products showed activity as inhibitors of Topoisomerase I (TopI). Additionally, the cytotoxic behavior of these compounds has been studied in cell lines derived from human lung adenocarcinoma (A549) and human ovarian carcinoma (SKOV03), and on non-cancerous lung fibroblasts cell line (MRC5) where, on the last ones, the absence of cytotoxicity was observed. 7-Phenyl-6H-6a,7,12,12a-tetrahydrochromeno[4,3-b][1,5]naphthyridine 5a showed excellent cytotoxic activity with a IC50 value of 1.03⯱â¯0.30⯵M against the A549â¯cell line and a IC50 value of 1.75⯱â¯0.20⯵M against the SKOV03â¯cell line. The obtained results point to these compounds as good antiproliferative candidates.
Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Naftiridinas/farmacologia , Inibidores da Topoisomerase I/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo I/isolamento & purificação , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Estrutura Molecular , Naftiridinas/síntese química , Naftiridinas/química , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/química , Células Tumorais CultivadasRESUMO
We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase É (Pol É), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.
Assuntos
DNA Polimerase Dirigida por DNA/análise , DNA Polimerase Dirigida por DNA/isolamento & purificação , Eletroforese/métodos , Complexos Multienzimáticos/análise , Complexos Multienzimáticos/isolamento & purificação , Antígenos Virais de Tumores/genética , Extratos Celulares/química , Linhagem Celular Tumoral , DNA Polimerase I/isolamento & purificação , DNA Polimerase II/isolamento & purificação , DNA Polimerase III/isolamento & purificação , Replicação do DNA , DNA Topoisomerases Tipo I/isolamento & purificação , Células HeLa , Humanos , Antígeno Nuclear de Célula em Proliferação/análise , Origem de Replicação/genética , Proteína de Replicação A/isolamento & purificação , Proteína de Replicação C/isolamento & purificação , Vírus 40 dos Símios/genéticaRESUMO
The different steps of the topoisomerase I catalytic cycle have been analyzed in the presence of the plant alkaloid thaspine (1- (2-(Dimethylamino)ethyl)-3,8-dimethoxychromeno[5,4,3-cde]chromene-5,10-dione), known to induce apoptosis in colon carcinoma cells. The experiments indicate that thaspine inhibits both the cleavage and the religation steps of the enzyme reaction. The inhibition is reversible and the effect is enhanced upon pre-incubation. Molecular docking simulations of thaspine over topoisomerase I, in the presence or absence of the DNA substrate, show that thaspine, when interacting with the enzyme alone in the closed or in the open state, can bind in proximity of the active residues preventing the cleavage reaction, whilst when docked with the enzyme-DNA cleavable complex intercalates between the DNA bases in a way similar to that found for camptothecin, explaining its religation inhibition. These results unequivocally demonstrate that thaspine targets human topoisomerase I .
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Inibidores da Topoisomerase I/farmacologia , Alcaloides/química , Antineoplásicos/química , Produtos Biológicos/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/isolamento & purificação , Humanos , Cinética , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/químicaRESUMO
DNA topoisomerases play critical roles in regulating DNA topology and are essential enzymes for cell survival. In this study, a gene encoding type IA DNA topoisomerase was cloned from Staphylococcus aureus (S. aureus) sp. strain C-66, and the biochemical properties of recombinant enzyme was characterized. The nucleotide sequence analysis showed that the cloned gene contained an open reading frame (2070 bp) that could encode a polypeptide of 689 amino acids. The cloned gene actually produced 79.1 kDa functional enzyme (named Sau-TopoI) in Escherichia coli (E. coli). Sau-TopoI enzyme purified from E. coli showed ATP-independent and Mg(2+)-dependent manners for relaxing negatively supercoiled DNA. The relaxation activity of Sau-TopoI was inhibited by camptothecin, but not by nalidixic acid and etoposide. Cleavage site mapping showed that the enzyme could preferentially bind to and cleave the sequence GGNN↓CAT (N and ↓ represent any nucleotide and cleavage site, respectively). All these results suggest that the purified enzyme is type IA DNA topoisomerase. In addition, domain mapping analysis showed that the enzyme was composed of conserved four domains (I through IV), together with a variable C-terminal region containing a unique domain V.
Assuntos
DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , Staphylococcus aureus/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cátions Bivalentes/metabolismo , Clonagem Molecular , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo I/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Temperatura , Fatores de Tempo , Inibidores da Topoisomerase I/farmacologiaRESUMO
Escherichia coli topoisomerase I (TopA) cleaves and rejoins one strand of double-stranded DNA to relax the negatively supercoiled DNA. Structurally, TopA contains an N-terminal catalytic fragment and a C-terminal zinc-binding region that is required for relaxation of the negatively supercoiled DNA. Here we report that E. coli TopA is an iron and zinc binding protein. The UV-Vis absorption measurements and metal content analyses reveal that TopA purified from E. coli cells grown in the rich LB medium contains both iron and zinc. However, TopA purified from E. coli cells grown in the M9 minimal medium has negligible amounts of zinc or iron and no topoisomerase activity. Nevertheless, supplement of exogenous zinc or iron in E. coli cells grown in the M9 minimal medium produces the zinc- or iron-bound TopA, respectively. Whereas the zinc-bound TopA is fully active to relax the negatively supercoiled DNA, the iron-bound TopA has little or no enzyme activity. Furthermore, excess iron in the M9 minimal medium is able to compete with the zinc binding in TopA in E. coli cells and attenuate the topoisomerase activity, suggesting that E. coli TopA may be modulated by iron and zinc binding in vivo.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Escherichia coli/enzimologia , Ferro/metabolismo , Zinco/metabolismo , Sítios de Ligação , DNA Topoisomerases Tipo I/isolamento & purificação , Escherichia coli/citologia , Espectrofotometria UltravioletaRESUMO
The use of co-immunoprecipitation (co-IP) to purify multi-protein complexes has contributed greatly to our understanding of the DNA damage response network associated with Fanconi anemia (FA), Bloom syndrome (BS) and breast cancer. Four new FA genes and two new protein partners for the Bloom syndrome gene product have been identified by co-IP. Here, we discuss our experience in using co-IP and other techniques to isolate and characterize new FA and BS-related proteins.
Assuntos
Síndrome de Bloom/genética , Proteínas de Transporte/análise , Dano ao DNA , DNA Topoisomerases Tipo I/análise , Proteínas de Ligação a DNA/análise , Anemia de Fanconi/genética , Proteínas Nucleares/análise , RecQ Helicases/análise , Síndrome de Bloom/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Anemia de Fanconi/metabolismo , Humanos , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , RecQ Helicases/isolamento & purificação , RecQ Helicases/metabolismoRESUMO
Reverse gyrases are topoisomerases that catalyze ATP-dependent positive supercoiling of circular covalently closed DNA. They consist of an N-terminal helicase-like domain, fused to a C-terminal topoisomerase I-like domain. Most of our knowledge on reverse gyrase-mediated positive DNA supercoiling is based on studies of archaeal enzymes. To identify general and individual properties of reverse gyrases, we set out to characterize the reverse gyrase from a hyperthermophilic eubacterium. Thermotoga maritima reverse gyrase relaxes negatively supercoiled DNA in the presence of ADP or the non-hydrolyzable ATP-analog ADPNP. Nucleotide binding is necessary, but not sufficient for the relaxation reaction. In the presence of ATP, positive supercoils are introduced at temperatures above 50 degrees C. However, ATP hydrolysis is stimulated by DNA already at 37 degrees C, suggesting that reverse gyrase is not frozen at this temperature, but capable of undergoing inter-domain communication. Positive supercoiling by reverse gyrase is strictly coupled to ATP hydrolysis. At the physiological temperature of 75 degrees C, reverse gyrase binds and hydrolyzes ATPgammaS. Surprisingly, ATPgammaS hydrolysis is stimulated by DNA, and efficiently promotes positive DNA supercoiling, demonstrating that inter-domain communication during positive supercoiling is fully functional with both ATP and ATPgammaS. These findings support a model for communication between helicase-like and topoisomerase domains in reverse gyrase, in which an ATP and DNA-induced closure of the cleft in the helicase-like domain initiates a cycle of conformational changes that leads to positive DNA supercoiling.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteínas de Bactérias/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Super-Helicoidal/metabolismo , Conformação de Ácido Nucleico , Thermotoga maritima , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Super-Helicoidal/química , Modelos Moleculares , Mutação , Nucleotídeos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermotoga maritima/enzimologia , Thermotoga maritima/genéticaRESUMO
The prostate-specific homeodomain protein NKX3.1 is a tumor suppressor that is commonly down-regulated in human prostate cancer. Using an NKX3.1 affinity column, we isolated topoisomerase I (Topo I) from a PC-3 prostate cancer cell extract. Topo I is a class 1B DNA-resolving enzyme that is ubiquitously expressed in higher organisms and many prokaryotes. NKX3.1 interacts with Topo I to enhance formation of the Topo I-DNA complex and to increase Topo I cleavage of DNA. The two proteins interacted in affinity pull-down experiments in the presence of either DNase or RNase. The NKX3.1 homeodomain was essential, but not sufficient, for the interaction with Topo I. NKX3.1 binding to Topo I occurred independently of the Topo I NH2-terminal domain. The binding of equimolar amounts of Topo I to NKX3.1 caused displacement of NKX3.1 from its cognate DNA recognition sequence. Topo I activity in prostates of Nkx3.1+/- and Nkx3.1-/- mice was reduced compared with wild-type mice, whereas Topo I activity in livers, where no NKX3.1 is expressed, was independent of Nkx3.1 genotype. Endogenous Topo I and NKX3.1 could be coimmunoprecipitated from LNCaP cells, where NKX3.1 and Topo I were found to colocalize in the nucleus and comigrate within the nucleus in response to either gamma-irradiation or mitomycin C exposure, two DNA-damaging agents. This is the first report that a homeodomain protein can modify the activity of Topo I and may have implications for organ-specific DNA replication, transcription, or DNA repair.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Proteínas de Homeodomínio/metabolismo , Neoplasias da Próstata/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Cromatografia de Afinidade/métodos , DNA Topoisomerases Tipo I/isolamento & purificação , DNA de Neoplasias/metabolismo , Ativação Enzimática , Humanos , Cinética , Masculino , Camundongos , Neoplasias da Próstata/enzimologia , Ligação ProteicaRESUMO
Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes dozens of proteins with imputed functions in nucleic acid transactions. Here we produced, purified, and characterized mimivirus DNA topoisomerase IB (TopIB), which we find to be a structural and functional homolog of poxvirus TopIB and the poxvirus-like topoisomerases discovered recently in bacteria. Arginine, histidine, and tyrosine side chains responsible for TopIB transesterification are conserved and essential in mimivirus TopIB. Moreover, mimivirus TopIB is capable of incising duplex DNA at the 5'-CCCTT cleavage site recognized by all poxvirus topoisomerases. Based on the available data, mimivirus TopIB appears functionally more akin to poxvirus TopIB than bacterial TopIB, despite its greater primary structure similarity to the bacterial TopIB group. We speculate that the ancestral bacterial/viral TopIB was disseminated by horizontal gene transfer within amoebae, which are permissive hosts for either intracellular growth or persistence of many present-day bacterial species that have a type IB topoisomerase.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Entomopoxvirinae/enzimologia , Transferência Genética Horizontal , Genoma Viral , Acanthamoeba/virologia , Sequência de Aminoácidos , Animais , Bactérias/genética , Bactérias/metabolismo , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Super-Helicoidal/metabolismo , Entomopoxvirinae/química , Entomopoxvirinae/genética , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
Poly(ADP-ribose) polymerase-1 (PARP-1) is known to have an important role in camptothecin sensitivity and interacts with topoisomerase I. In the present study, the impact of PARP-1 on the topoisomerase I-DNA complex stabilized by camptothecin was assessed. It was shown that NH2 terminus-truncated topoisomerase I (amino acids 201-765) showed at least 4-fold less sensitivity to camptothecin than full-length topoisomerase I in the oligonucleotide religation assay. PARP-1 could prevent the action of camptothecin on the religation activity of full-length topoisomerase I, which is linked to DNA in a stoichiometrical manner. However, the religation activity of NH2 terminus-truncated topoisomerase I, which is linked to DNA, could not be enhanced by PARP-1 in the presence of camptothecin. Both full-length and NH2 terminus-truncated topoisomerase I interact with PARP-1. This data suggests that PARP-1 destabilizes the topoisomerase I-camptothecin-DNA complex with the participation of the NH2-terminal domain of topoisomerase I. Poly(ADP-ribosyl)ation of topoisomerase I by PARP-1 in the presence its substrate, NAD, could also promote the religation activity of full-length topoisomerase I as well as NH2 terminus-truncated topoisomerase I. PARP-1 inhibitors (3-aminobenzamide, PJ34) could inhibit this process. Therefore, PARP-1 could facilitate the religation activity of topoisomerase I by itself through topoisomerase I-PARP-1 interaction (PARP-1 action) or by the formation of poly(ADP-ribosyl)ation of topoisomerase I (PARP-1/NAD action). This study also implies that PARP-1 and PARP-1/NAD actions need to be highly regulated by cellular factors for camptothecin to exert its cytotoxicity inside the cells. We propose ATP to be one of the important regulatory factors.
Assuntos
Camptotecina/farmacologia , DNA/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores da Topoisomerase I , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , DNA/antagonistas & inibidores , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo I/metabolismo , Humanos , NAD/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph.
Assuntos
DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo I/metabolismo , Piscirickettsiaceae/classificação , Piscirickettsiaceae/enzimologia , Trifosfato de Adenosina/farmacologia , Cátions Bivalentes/farmacologia , Cromatografia Líquida de Alta Pressão , DNA Topoisomerases Tipo I/classificação , DNA de Cadeia Simples/farmacologia , Ácido Edético/farmacologia , Etídio/farmacologia , Concentração de Íons de Hidrogênio , Manganês/farmacologia , Biologia Marinha , Fatores de Tempo , Inibidores da Topoisomerase IRESUMO
DNA topoisomerases (topo) are the cellular targets of several anticancer drugs used today in the clinic. Our previous work demonstrated that certain tyrphostin derivatives, known as protein tyrosine kinase antagonists, are catalytic inhibitors of DNA topoisomerases I (topo I) in vitro. In this study, we examined the ability of tyrphostin derivatives to affect the activity of topo I in the cell (in vivo) and determined their in vivo mode of action. Two approaches were used; in the first, we examined the direct effect of the treatment of tumor cells with tyrphostins on the activity, level, and post-translational modifications of the cellular topo I. The second approach was to determine the influence of pretreatment of tumor cells with tyrphostin on the cellular induced effects of camptothecin (CPT), a known inhibitor of topo I. The results show that treatment of fibrosarcoma cells with tyrphostin inhibited the DNA relaxation activity of topo I but did not reduce the level of topo I protein. Tyrphostin treatments caused conformational changes of the cellular topo I probably by binding to the enzyme. Pretreatment of the cells with tyrphostin before CPT prevented the CPT-induced degradation of topo I and reduced the enzyme-DNA cleavable complexes and the ubiquitination/sumoylation of the enzyme. These data suggest that topo I is one of the cellular targets of tyrphostin and that this drug acts in vivo (in the cell) as a catalytic inhibitor of the enzyme that alters the binding of the enzyme to the DNA.
Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Expressão Gênica/efeitos dos fármacos , Tirfostinas/farmacologia , Células 3T3 , Animais , Camptotecina/farmacologia , Extratos Celulares/farmacologia , DNA/efeitos dos fármacos , DNA/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/isolamento & purificação , Camundongos , Conformação Proteica , Proteína SUMO-1/metabolismo , Inibidores da Topoisomerase I , Células Tumorais Cultivadas , Tirfostinas/química , Ubiquitina/metabolismoRESUMO
In cocrystal structures of human topoisomerase I and DNA, the enzyme is tightly clamped around the DNA helix. After cleavage and covalent attachment of the enzyme to the 3' end at the nick, DNA relaxation requires rotation of the DNA helix downstream of the cleavage site. Models based on the cocrystal structure reveal that there is insufficient space in the protein for such DNA rotation without some deformation of the cap and linker regions of the enzyme. Alternatively, it is conceivable that the protein clamp opens to facilitate the rotation process. To distinguish between these two possibilities, we engineered two cysteines into the opposing loops of the "lips" region of the enzyme, which allowed us to lock the protein via a disulfide crosslink in the closed conformation around the DNA. Importantly, the rate of DNA relaxation when the enzyme was locked on the DNA was comparable to that observed in the absence of the disulfide crosslink. These results indicate that DNA relaxation likely proceeds without extensive opening of the enzyme clamp.
Assuntos
DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Substituição de Aminoácidos , Centrifugação com Gradiente de Concentração , DNA/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/isolamento & purificação , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Plasmídeos , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
PURPOSE: Peganum harmala L. (Zygophyllaceae) seeds extract is one of the main components of an ethnobotanical preparation used in the treatment of neoplasms in Iran. Cytotoxic effects of P. harmala extract on cancerous cell-lines have been reported before. beta-carbolines like harmaline and harmine are the major alkaloids present in the seeds of the P. harmala. Considering reports concerning DNA topoisomerase inhibition by other beta-carbolines like harmane, we have used DNA relaxation assays to investigate topoisomerase I inhibitory activity of P. harmala seeds extract and its beta-carboline alkaloids to further inspecting the mechanism of its cytotoxic activity. METHODS: Harmine and harmaline contents of the extract were determined using an HPTLC method. DNA topoisomerase I enzyme needed for investigating inhibitory effect of the compounds using DNA relaxation assay, was partially purified from the human placenta. DNA relaxation assay is based on the conversion of a supercoiled plasmid substrate to its relaxed form by the catalytic activity of the enzyme. The supercoiled substrate and its relaxed product can be easily distinguished using agarose gel electrophoresis, since the relaxed topological isomers of DNA migrate more slowly than supercoiled species. RESULTS: Using HPTLC method, it was found that each gram of dried extract contained 55.5 and 79.0 mg of harmine and harmaline respectively. In the DNA relaxation assay, order of potency was harmine > harmane > harmaline > extract. The most active compound was harmine with IC50 value of 13.5 +/- 1.7 microg/ml. CONCLUSIONS: Our in vitro findings demonstrate that P. harmala seeds extract do inhibit human DNA topoisomerase I and based on the results of HPTLC analysis, it appears that the biological activity of the extract can be explained by its beta-carboline content.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Extratos Vegetais/farmacologia , Inibidores da Topoisomerase I , Zygophyllaceae/química , Cromatografia Líquida de Alta Pressão/métodos , DNA/metabolismo , DNA Topoisomerases Tipo I/isolamento & purificação , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Placenta/enzimologia , Sementes/químicaRESUMO
All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, strand rotation, and ligation. A comparison with previously published studies showing no effect of deleting the first 174 or 190 amino acids of topoisomerase I (Stewart, L., Ireton, G. C., and Champoux, J. J. (1999) J. Biol. Chem. 274, 32950-32960; Bronstein, I. B., Wynne-Jones, A., Sukhanova, A., Fleury, F., Ianoul, A., Holden, J. A., Alix, A. J., Dodson, G. G., Jardillier, J. C., Nabiev, I., and Wilkinson, A. J. (1999) Anticancer Res. 19, 317-327) suggests a pivotal role of amino acids 191-206 in catalysis. Taken together the presented data indicate that at least part(s) of the N-terminal domain regulate(s) enzyme/DNA dynamics during relaxation most probably by controlling non-covalent DNA binding downstream of the cleavage site either directly or by coordinating DNA contacts by other parts of the enzyme.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Catálise , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Humanos , Conformação ProteicaRESUMO
Poly(ADP-ribose) polymerase (PARP I) and Topoisomerase I (Topo I) were reisolated from calf thymus to eliminate cross contamination as tested by immunotransblots. The specific activity of Topo I was greatly increased by added PARP I, following saturation kinetics. Recombinant PARP I and isolated PARP I at final purity were indistinguishable in terms of their activation of Topo I. There was a coincidence of experimentally obtained binding constants and computer generated values based on the kinetic model, indicating that the association of PARP I and Topo I is rate limiting in the catalytic activation of Topo I by PARP I. Polypeptide domains of PARP I that are required for protein-protein binding and protein-DNA binding also activate Topo I. Fluorescence resonance energy transfer between fluorophor-labeled PARP I and Topo I was demonstrated. The binding of Topo I to circular SV40 DNA, assayed either by the formation of a) the sum of non-covalently and covalently attached Topo I to DNA or b) by the covalently bound transient intermediate in the presence of camptothecin, was augmented when PARP I protein was bound to SV40 DNA. These binding experiments provide a molecular basis for the kinetic activation of Topo I by PARP I inasmuch as the increased superhelicity of SV40 DNA induced by PARP I may facilitate the formation of a more
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Bovinos , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Circular/metabolismo , Transferência de Energia , Ativação Enzimática , Fluorescência , Cinética , NAD/metabolismo , Fragmentos de Peptídeos/metabolismo , Poli(ADP-Ribose) Polimerases/isolamento & purificaçãoRESUMO
Melanoplus sanguinipes entomopoxvirus (MsEPV) encodes a 328 amino acid polypeptide related to the type I topoisomerases of six other genera of vertebrate and insect poxviruses. The gene encoding MsEPV topoisomerase was expressed in bacteria, and the recombinant protein was purified by ion-exchange chromatography and glycerol gradient sedimentation. MsEPV topoisomerase, a monomeric protein, catalyzed the relaxation of supercoiled plasmid DNA at approximately 0.6 supercoils/s. Like other poxvirus topoisomerases, the MsEPV enzyme formed a covalent adduct with duplex DNA at the target sequence CCCTT downward arrow. The kinetic and equilibrium parameters of the DNA transesterification reaction of MsEPV topoisomerase were k(cl) = 0.3 s(-1) and K(cl) = 0.25. The introduction of a 5'-bridging phosphorothiolate at the scissile phosphate increased the cleavage equilibrium constant from 0.25 to >/=30. Similar phosphorothiolate effects were observed with vaccinia topoisomerase. Kinetic analysis of single-turnover cleavage and religation reactions established that the altered equilibrium was the result of a approximately 10(-4) decrement in the rate of topoisomerase-catalyzed attack of 5'-SH DNA on the DNA-(3'-phosphotyrosyl)-enzyme intermediate. 5'-bridging phosphorothiolates at the scissile phosphate and other positions within the CCCTT element had no significant effect on k(cl).
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Entomopoxvirinae/enzimologia , Gafanhotos/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/isolamento & purificação , DNA de Cadeia Simples , Esterificação , Expressão Gênica , Dados de Sequência Molecular , Fosfatos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Tionucleotídeos/metabolismoRESUMO
Traditional models for transcription initiation by RNA polymerase I include a stepwise assembly of basic transcription factors/regulatory proteins on the core promoter to form a preinitiation complex. In contrast, we have identified a preassembled RNA polymerase I (RPI) complex that contains all the factors necessary and sufficient to initiate transcription from the rDNA promoter in vitro. The purified RPI holoenzyme contains the RPI homolog of TFIID, SL-1 and the rDNA transcription terminator factor (TTF-1), but lacks UBF, an activator of rDNA transcription. Certain components of the DNA repair/replication system, including Ku70/80, DNA topoisomerase I and PCNA, are also associated with the RPI complex. We have found that the holo-enzyme supported specific transcription and that specific transcription was stimulated by the RPI transcription activator UBF. These results support the hypothesis that a fraction of the RPI exists as a preassembled, transcriptionally competent complex that is readily recruited to the rDNA promoter, i.e. as a holoenzyme, and provide important new insights into the mechanisms governing initiation by RPI.
Assuntos
Antígenos Nucleares , DNA Helicases , Reparo do DNA , Replicação do DNA , Complexos Multienzimáticos/química , Proteínas Pol1 do Complexo de Iniciação de Transcrição , RNA Polimerase I/química , RNA Polimerase I/isolamento & purificação , Fatores de Transcrição/isolamento & purificação , Animais , Reparo do DNA/genética , Replicação do DNA/genética , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo I/metabolismo , DNA Ribossômico/genética , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Holoenzimas/química , Holoenzimas/isolamento & purificação , Holoenzimas/metabolismo , Autoantígeno Ku , Peso Molecular , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/isolamento & purificação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Polimerase I/metabolismo , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/análise , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Células Tumorais CultivadasRESUMO
Human DNA topoisomerase I (topo I) has been purified from normal placenta and from a recombinant baculovirus expression system. A new radiolabeled plasmid DNA assay has been used to quantitate the activity of the purified enzymes and to compare the ability of several types of topo I-targeted drugs to induce topo I-mediated DNA strand breaks. The 100-kDa recombinant enzyme form isolated from the baculovirus expression system is able to relax 2564 ng of supercoiled M-13 mp19 plasmid per minute per nanogram of enzyme. The addition of camptothecin (1 microM) to the reaction lowers the rate to 1282 ng per minute per nanogram of enzyme. The 100-kDa topo I from human placenta is able to relax 1092 ng of supercoiled plasmid per minute per nanogram of enzyme and the 68-kDa topo I form from placenta is able to relax 2069 ng of supercoiled plasmid per minute per nanogram of enzyme. Camptothecin (1 microM) decreases the relaxation rate of the placental enzymes about 50%. In the presence of several different types of topo I-targeted drugs, both the recombinant and placental enzymes are induced to cleave plasmid DNA. Quantitative DNA cleavage assays with radioactive plasmid DNA and 9-aminocamptothecin, topotecan, SN-38, 10, 11-methylenedioxycamptothecin, 7-ethyl-10, 11-methylenedioxycamptothecin, 7-chloromethyl-10, 11-methylenedioxycamptothecin, nitidine, and 6-ethoxy-5, 6-dihydronitidine indicate that the order of potency in inducing topo I-mediated DNA breakage is methylenedioxycamptothecin analogs > SN-38 > 9-aminocamptothecin > topotecan and camptothecin > nitidine compounds. The order of potency correlates with the half-lives of the topo I-DNA drug complex determined with radiolabeled DNA in 0.45 M NaCl at 30 degrees C. The half-life of the complex formed with 7-chloromethyl-10,11-methylenedioxycamptothecin is greater than 90 min whereas the half-life of the topo I-DNA complex with 6-ethoxy-5, 6-dihydronitidine is less than 15 s. The other drugs tested were found to have drug complex half-lives which fall between these two extremes.
Assuntos
Camptotecina/análogos & derivados , Camptotecina/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Fenantridinas/farmacologia , Animais , Antineoplásicos/farmacologia , Benzofenantridinas , Linhagem Celular , DNA Topoisomerases Tipo I/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Cinética , Placenta/enzimologia , Plasmídeos/metabolismo , Gravidez , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecçãoRESUMO
DNA topoisomerase I is a nuclear enzyme involved in transcription, recombination, and DNA damage recognition. Previous studies have shown that topoisomerase I interacts directly with the tumor-suppressor protein p53. p53 is a transcription factor that activates certain genes through binding to specific DNA sequences. We now report that topoisomerase I can be stimulated by both latent and activated wild-type p53 as well as by several mutant and truncated p53 proteins in vitro, indicating that sequence-specific DNA-binding and stimulation of topoisomerase I are distinct properties of p53. These assays also suggest that the binding site for topoisomerase I on p53 is between amino acids 302 and 321. In living cells, the interaction between p53 and topoisomerase I is strongly dependent on p53 status. In MCF-7 cells, which have wild-type p53, the association between the two proteins is tightly regulated in a spatial and temporal manner and takes place only during brief periods of genotoxic stress. In marked contrast, the two proteins are constitutively associated in HT-29 cells, which have mutant p53. These findings have important implications for both cellular stress response and genomic stability, given the ability of topoisomerase I to recognize DNA lesions as well as to cause illegitimate recombination.