RESUMO
Eukaryotic topoisomerase I polypeptides can be partitioned into four structural domains. The function of the N-terminal domain, which is a target for serine-specific phosphorylation, has not been fully defined. The number of serine residues in the N-terminal domain of topoisomerase I from different species is inversely proportional to the number of charged amino acids in this region of the protein. The significance of this correlation is discussed in terms of a possible role for serine-specific phosphorylation in the activity of the enzyme.
Assuntos
DNA Topoisomerases Tipo I/química , Fosfosserina/análise , Animais , Caseína Quinase II , Cromatografia em Camada Fina , Sequência Consenso , DNA Topoisomerases Tipo I/metabolismo , Células Eucarióticas/enzimologia , Humanos , Ponto Isoelétrico , Mapeamento de Peptídeos , Fosforilação , Fosfosserina/metabolismo , Proteína Quinase C , Proteínas Serina-Treonina Quinases , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Serina/análise , TripsinaRESUMO
An in vitro system composed of nicked pBR322 DNA and purified topoisomerase I was employed to study the efficiency of the topoisomerase I-driven single-strand to double-strand DNA breaks conversion. At 1.4 x 10(5) topoisomerase I activity units per mg DNA about 20% single-strand nicks were converted into double-strand breaks during 30 min due to topoisomerase I action. Camptothecin inhibited the conversion. The conversion was also inhibited when the relaxing activity of the used topoisomerase I was increased by phosphorylation of the enzyme with casein kinase 2. The presented data suggest that topoisomerase I may be involved in production of double-stranded breaks in irradiated cells and that this process positively depends on the amount of topoisomerase I but not on its phosphorylation state.
Assuntos
Dano ao DNA , DNA Topoisomerases Tipo I/metabolismo , DNA de Cadeia Simples/metabolismo , Animais , Camptotecina/farmacologia , Caseína Quinase II , Bovinos , DNA/metabolismo , DNA/efeitos da radiação , DNA Super-Helicoidal/metabolismo , Raios gama , Fosforilação , Plasmídeos/metabolismo , Plasmídeos/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
cDNA encoding DNA topoisomerase I from Physarum polycephalum was isolated from a poly(A)+ -primed library (3'-region) and by PCR (5'-region). The coding region of cDNA was 3045 bp, encoding a polypeptide of molecular mass of 112 kDa. Identity between predicted amino acids sequences of conserved domains and corresponding domains from another eukaryotic type I DNA topoisomerases varied from 33.2 to 53.5% for the core domain and from 33.8 to 57.4% for the C-terminal domain. A peculiar feature of Physarum DNA topoisomerase I was a stretch of repeated KPAX...X motifs in the N-terminal domain of the polypeptide. Although treatment of the plasmodia with db-cAMP increased relaxing activity of the DNA topoisomerase I several-fold, there was only a slight increase in the mRNA level.
Assuntos
Bucladesina/farmacologia , DNA Topoisomerases Tipo I/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Physarum polycephalum/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem de Organismos , Sequência Conservada , AMP Cíclico/metabolismo , DNA Topoisomerases Tipo I/química , DNA Complementar , Dados de Sequência Molecular , Peso Molecular , Physarum polycephalum/genética , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tetrahymena/genéticaRESUMO
Relaxing activity of Physarum topoisomerase I was increased by calf thymus protein kinase ck2, similarly as was the activity of mammalian topoisomerase I, despite a pronounced difference between amino-acid sequences of non-conserved domains of Physarum and mammalian enzymes. This feature of Physarum topoisomerase I was cancelled in nuclear extracts isolated from dibutyryl-cAMP treated plasmodia in which the activity of protein kinase ck2 was elevated.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Physarum polycephalum/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Bucladesina/farmacologia , Caseína Quinase II , Bovinos , DNA Topoisomerases Tipo I/química , Cinética , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Timo/enzimologiaAssuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Leucemia L5178/enzimologia , Animais , Resistência a Medicamentos , Leucemia L5178/patologia , Camundongos , Fosforilação , Poli Adenosina Difosfato Ribose/metabolismo , Células Tumorais CultivadasRESUMO
The reason for different phosphorylation of topoisomerase I in two sublines of L5178Y murine lymphoma (LY cells) was investigated. Camptothecin-resistant LY-S cells show increased poly(ADP-ribose) level and lowered topoisomerase I phosphorylation compared to camptothecin-sensitive LY-R cells. In this study diminished phosphorylation of LY-S topoisomerase I was observed for sites recognized by casein kinase 2 but not for those phosphorylated by protein kinase C. Tryptic digests of LY-S topoisomerase I labeled in vitro by casein kinase 2 indicated that phosphorylation was similarly lowered at different sites. Activity of casein kinase 2 measured in nuclear extracts was about 1.7 times lower for LY-S than LY-R cells. This difference was diminished or eliminated by increasing casein concentration, diluting the extract or increasing the ionic strength. Activity of poly(ADP-ribose) polymerase was 5.3 times higher in LY-S than in LY-R nuclei. When the activity of the polymerase was inhibited by treatment of LY-S cells with benzamide, casein kinase 2-catalyzed phosphorylation of topoisomerase I increased. This was accompanied by an increase in sensitivity to camptothecin as reflected in the diminished viability of LY-S cells.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Leucemia L5178/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Benzamidas/farmacologia , Camptotecina/farmacologia , Caseína Quinase II , Interações Medicamentosas , Camundongos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Two sublines of LY murine lymphoma, differing in sensitivity to CPT, served as source of topoisomerase I in order to compare the enzyme's properties. The activity of topoisomerase I isolated from LY-S cells of reduced sensitivity to CPT increased about 2-times more upon phosphorylation with casein kinase but was inhibited to a lesser extent upon dephosphorylation with alkaline phosphatase than the enzyme from the CPT-sensitive LY-R cells. The in vitro phosphorylation of LY-S enzyme restored its sensitivity to CPT. The in vitro incorporation of 32P into topoisomerase protein was about 1.7-times higher in LY-S than in LY-R enzyme. A reversed incorporation ratio was observed upon metabolic labelling. The level of topoisomerase I protein, determined by Western blot analysis using scleroderma anti-topoisomerase I antibodies, was about 1.5-times higher in LY-S than in LY-R cells. The level of topoisomerase I mRNA was similar in both sublines. These results indicate that the reduced sensitivity of LY-S cells to CPT is based on the lowered phosphorylation of topoisomerase I protein but does not depend on the expression of topoisomerase I gene.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Animais , DNA Topoisomerases Tipo I/genética , Linfoma/enzimologia , Camundongos , Fosforilação , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Sensitivity to camptothecin (CPT) of type I DNA topoisomerases isolated from two L5178Y (LY) sublines was examined in reaction media containing either aspartate or chloride. The enzyme from LY-S cells was sensitive to the drug in the presence of 120 mM K-aspartate, but the sensitivity was markedly reduced in the presence of 120 mM KCl. The enzyme from LY-R cells was similarly sensitive to camptothecin in the presence of either aspartate or chloride. The optimum ionic strength for the relaxation reaction catalyzed by both LY-R and LY-S type I DNA topoisomerases was similar. We suggest that sensitivity of the LY-S enzyme to CPT depends on the amount of cleavable complex formed, which in turn depends on the ionic conditions of the assay.
Assuntos
Camptotecina/farmacologia , Leucemia L5178/enzimologia , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores da Topoisomerase I , Animais , Ácido Aspártico/farmacologia , Resistência a Medicamentos , Camundongos , Cloreto de Potássio/farmacologia , Células Tumorais CultivadasRESUMO
The level of topoisomerase I mRNA was measured in cells of two mouse lymphoma (LY) sublines treated with db-cAMP. A transient increase of the level was observed to be of about 60% of the basic level and to have maximum after the 3 h treatment of LY-S cells. The increase in LY-R subline was two-fold lower. The activity of PKA in a cytosol fraction of LY-S cells was 1.75 times higher than that in LY-R cells. The activity of PKA in membranes and nuclear fraction did not differ significantly in both cell types. When the activity of PKA in LY-S cells was inhibited with H8, no increase of the level of topoisomerase I mRNA was observed upon db-cAMP treatment of cells. We suggest that the activity of PKA in the cytosol controls the expression of topoisomerase I gene in LY cells at high concentration of cAMP.
Assuntos
Bucladesina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA Topoisomerases Tipo I/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Animais , Compartimento Celular , Citosol/enzimologia , DNA Topoisomerases Tipo I/genética , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia L5178/enzimologia , Camundongos , RNA Mensageiro/biossínteseRESUMO
Murine L517BY (LY) lymphoma sublines, LY-R (X-radiation resistant) and LY-S (X-radiation sensitive) displayed a difference in susceptibility to camptothecin: susceptibility of LY-S cells to the alkaloid was shifted towards higher concentrations as compared to LY-R cells. A similar difference was observed at the level of genomic DNA when a number of DNA-protein cross-links was determined or single-strand breaks were revealed by the fluorescent nucleoid halo assay. Activities of topoisomerases I and II were the same in both sublines. In turn, a higher resistance to camptothecin was found for the isolated LY-S topoisomerase I in the DNA cleavage test, suggesting that an altered enzyme was responsible for the susceptibility difference observed at the cellular level. In the relaxation test the enzymes from the two sublines showed a different sensitivity to beta-lapachone, an activator of topoisomerase I, but were similarly sensitive to all inhibitors, except camptothecin.
Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Leucemia L5178/enzimologia , Animais , Antibióticos Antineoplásicos/farmacologia , Núcleo Celular/enzimologia , Sobrevivência Celular/efeitos da radiação , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo II/metabolismo , Cinética , Camundongos , Naftoquinonas/farmacologia , Plasmídeos , Especificidade por Substrato , Inibidores da Topoisomerase I , Células Tumorais Cultivadas , Raios XRESUMO
Sensitivity to topoisomerase II inhibitors was tested at the cellular and enzyme level for two strains of mouse L5178Y lymphoma cells: resistant (LY-R) and sensitive (LY-S) to X-radiation. Differences in the susceptibility to inhibitors between LY-R and LY-S cells depended on the inhibitor used and were observed for adriamycin and VP-16, but not for mitoxantrone. On the other hand, isolated enzymes displayed the same sensitivity to all inhibitors tested regardless of the cell line. These results exclude the presence of altered topoisomerase II in LY-S cells as a possible reason for the collateral sensitivity of LY-S cells to X-radiation and topoisomerase II inhibitors.
Assuntos
Tolerância a Radiação , Inibidores da Topoisomerase II , Animais , DNA Topoisomerases Tipo II/metabolismo , Linfoma , Camundongos , Células Tumorais CultivadasRESUMO
1. A regulatory coupling between the rate of cellular transcription and the activity of topoisomerase I was investigated in plasmodia of Physarum polycephalum treated with fluorodeoxyuridine or nalidixic acid. 2. Fluorodeoxyuridine at concentrations above 40 micrograms/ml lowered both the incorporation of [3H]uridine and the activity of topoisomerase I to 10% of corresponding control values. 3. Nalidixic acid, in the range of concentrations between 20-50 micrograms/ml did not inhibit the incorporation of [3H]uridine but lowered the activity of topoisomerase I by about half. 4. It is suggested that a coupling between the level of transcription and the activity of topoisomerase I in Physarum plasmodia involves only about a half of the topoisomerase I activity and is limited to transcription occurring on ribosomal genes.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Floxuridina/farmacologia , Ácido Nalidíxico/farmacologia , Physarum polycephalum/enzimologia , Animais , Cinética , Physarum polycephalum/efeitos dos fármacos , RNA/biossíntese , Uridina/metabolismoRESUMO
A type I topoisomerase has been purified from nuclei of a slime mold Physarum polycephalum and its activity was tested during spherulation. The final preparation contained a single polypeptide of about 100 kDa. Basic properties of Physarum topoisomerase I (substrate specificity, ionic requirement, sensitivity to inhibitors) were similar to those of topoisomerases from higher eukaryotes. Specific features of Physarum enzyme were that it was rapidly inactivated at 45 degrees C and did not react with antibodies against human topoisomerase I. The activity of topoisomerase I in developed dormant spherules decreased approx. 2-fold, as compared with a 4-fold decrease of RNA and a 10-fold decrease of DNA synthesis. Basic properties of the enzyme remained unchanged during spherulation.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Physarum/enzimologia , DNA Fúngico/genética , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Imunoglobulina G/metabolismo , Physarum/crescimento & desenvolvimento , Especificidade por Substrato , TemperaturaAssuntos
Cromatina/análise , Cromossomos/ultraestrutura , Mitose , Acetilação , Animais , Cromossomos/metabolismo , Humanos , Fosforilação OxidativaRESUMO
The proteins of nuclear matrix preparations from Physarum polycephalum were compared with analogous mammalian fractions by gel electrophoresis, DNA-binding studies and immunological tests. Polypeptides of 28 and 36 K dalton, which dominate in Physarum preparations, differed from calf thymus matrix proteins in that they were basic and showed low affinity to DNA. These polypeptides were present at about 1.2 mg per mg of nuclear DNA. Polypeptides of higher molecular weight occurred in the preparation at about 0.5 mg per mg of nuclear DNA. At least some of the latter proteins showed high affinity to DNA and cross-reacted with the antiserum against calf thymus matrix proteins.
Assuntos
Núcleo Celular/análise , Proteínas Fúngicas/análise , Nucleoproteínas/análise , Physarum/análise , Antígenos Nucleares , Núcleo Celular/ultraestrutura , Imunodifusão , Ponto Isoelétrico , Peso MolecularRESUMO
Calf thymus chromatin gel, containing strongly bound nonhistone proteins, was used to study the effect of easily removable and tightly bound cations on the condensation of chromatin. The chromatin volume was found to be linearly dependent on the reciprocal square root of the concentration of easily removable cations (Tris X H+ + Na+ and Mg2+) except for the initial stages of condensation (up to 7-10 mM monovalent and 0.15-0.2 mM divalent cations). The effect of Mg2+ at the initial stage of condensation was not reproduced by Na+ and vice versa. At higher concentrations the effects of Na+ and Mg2+ were additive. The removal of tightly bound divalent cations by a treatment of the chromatin gel with 1,10-phenanthroline led to an approx. 50% increase in the volume of the chromatin gel, which was maintained at each concentration of easily removable cations. The 1,10-phenanthroline-caused decondensation of the chromatin gel was reversed by Ca2+ but not by Mg2+, Zn2+ and Cu2+. The chromatin gel pretreated with Ca2+ was not further decondensed by 1,10-phenanthroline.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Magnésio/farmacologia , Sódio/farmacologia , Animais , Cálcio/farmacologia , Bovinos , Cobre/farmacologia , Eletroforese em Gel de Poliacrilamida , Fenantrolinas/farmacologia , Zinco/farmacologiaAssuntos
Cromatina , Histonas , Mitose , DNA , Magnésio , Fosforilação , Protamina Quinase/metabolismoAssuntos
Cromatina/genética , Magnésio/fisiologia , Mitose , Modelos Genéticos , DNA , Conformação de Ácido Nucleico , ÁguaRESUMO
Chromatin of lower eukaryote Physarum polycephalum, while showing typical nucleosomal organization, reveals upon digestion with micrococcal nuclease certain features not found in chromatins of higher eukaryotes, the most pronounced of which is the unusual pattern of degradation of core-size DNA, without accumulation of subcore fragments. It has been shown that these peculiarities are not due to intrinsic features of Physarum nucleohistone complex but to the presence of a specific polysaccharide, the main component of Physarum slime, contaminating chromatin preparations.