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1.
Mol Plant Microbe Interact ; 30(2): 87-100, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27992291

RESUMEN

To elucidate one or more mechanisms through which microrchidia (MORC) proteins impact immunity, epigenetic gene silencing, and DNA modifications, the enzymatic activities of plant MORCs were characterized. Previously, we showed that plant MORC1s have ATPase and DNA endonuclease activities. Here, we demonstrate that plant MORCs have topoisomerase type II (topo II)-like activities, as they i) covalently bind DNA, ii) exhibit DNA-stimulated ATPase activity, iii) relax or nick supercoiled DNA, iv) catenate DNA, and v) decatenante kinetoplast DNA. Mutational analysis of tomato SlMORC1 suggests that a K loop-like sequence is required to couple DNA binding to ATPase stimulation as well as for efficient SlMORC1's DNA relaxation and catenation activities and in planta suppression of INF1-induced cell death, which is related to immunity. Human MORCs were found to exhibit the same topo II-like DNA modification activities as their plant counterparts. In contrast to typical topo IIs, SlMORC1 appears to require one or more accessory factors to complete some of its enzymatic activities, since addition of tomato extracts were needed for ATP-dependent, efficient conversion of supercoiled DNA to nicked/relaxed DNA and catenanes and for formation of topoisomer intermediates. Both plant and human MORCs bind salicylic acid; this suppresses their decatenation but not relaxation activity.


Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , ADN Superhelicoidal/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Plantas/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Biocatálisis , ADN/metabolismo , Humanos , Hidrólisis , Lisina/metabolismo , Mutación/genética , Proteínas Nucleares/química , Extractos Vegetales/metabolismo , Proteínas de Plantas/química , Unión Proteica , Ácido Salicílico/metabolismo
2.
J Plant Physiol ; 170(5): 497-504, 2013 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-23273927

RESUMEN

The translationally controlled tumor protein (TCTP) is a multi-functioning protein that carries out vital roles in various life processes. In this study, a new TCTP gene, designated as HbTCTP1, was isolated in Hevea brasiliensis. The full-length complementary DNA (cDNA) of HbTCTP1 contained a maximum open reading frame (ORF) of 507base pair (bp) encoding 168 amino acids. The sequence comparison showed that the deduced HbTCTP1 indicated high identities to plant TCTP proteins, and clustered in the dicot cluster of plant TCTPs. Although HbTCTP1 and human TCTP proteins did not parallel in overall sequence similarity, they indicated highly similar 3D structures with a nearly identical spatial organization of α-helices, ß-sheets, and coil regions. Real time reverse-transcription PCR (RT-PCR) analyses showed that HbTCTP1 was expressed throughout different tissues and developmental stages of leaves. Besides being related to tapping panel dryness (TPD), the HbTCTP1 transcripts were regulated by various treatments, including drought, low temperature, high salt, ethrel (ET), wounding, H2O2, and methyl jasmonate (Me-JA) treatments. The recombinant HbTCTP1 fusion protein was shown to protect supercoiled plasmid DNA from damages induced by metal-catalyzed generation of reactive oxygen species. The (45)Ca(2+)-overlay assay showed that HbTCTP1 was a calcium-binding protein. Our results are greatly helpful in understanding the molecular characterization and expression profiles of HbTCTP1, and lay the foundation for further analyzing the function of HbTCTP1 in rubber tree.


Asunto(s)
Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Hevea/genética , Goma/metabolismo , Secuencia de Bases , Biomarcadores de Tumor/química , Biomarcadores de Tumor/metabolismo , Proteínas de Unión al Calcio/metabolismo , Clonación Molecular , ADN Complementario/genética , ADN Superhelicoidal/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Proteína Tumoral Controlada Traslacionalmente 1
3.
Molecules ; 14(4): 1342-52, 2009 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-19384267

RESUMEN

Evodiamine (EVO), an alkaloidal compound isolated from Evodia rutaecarpa (Juss.), has been reported to affect many physiological functions. Topoisomerase inhibitors have been developed in a variety of clinical applications. In the present study, we report the topoisomerase I (TopI) inhibitory activity of EVO, which may have properties that lead to improved therapeutic benefits. EVO is able to inhibit supercoiled plasmid DNA relaxation catalyzed by TopI. Upon treatment 0-10 microM EVO TopI was depleted in MCF-7 breast cancer cells in a concentration-dependent and time-dependent manner in 0-120 min. A K-SDS precipitation assay was performed to measure the extent of Top I-trapped chromosomal DNA. The ability of EVO to cause the formation of a TopI-DNA complex increased in a concentration-dependent manner, in that the DNA trapped increased by 24.2% in cells treated with 30 microM. The results suggest that EVO inhibits TopI by stabilizing the enzyme and DNA covalent complex.


Asunto(s)
ADN Superhelicoidal/metabolismo , Sustancias Macromoleculares/metabolismo , Extractos Vegetales/metabolismo , Quinazolinas/metabolismo , Inhibidores de Topoisomerasa I , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo I/metabolismo , Estabilidad de Enzimas , Femenino , Humanos , Estructura Molecular , Extractos Vegetales/química , Quinazolinas/química
4.
Nucleic Acids Res ; 36(17): 5516-29, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18723572

RESUMEN

Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (-1 position). Mutagenesis of -1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable -1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.


Asunto(s)
Antibacterianos/toxicidad , Daño del ADN , Inhibidores Enzimáticos/toxicidad , Inhibidores de Topoisomerasa II , Adenosina Trifosfato/metabolismo , Antibacterianos/química , Secuencia de Bases , Girasa de ADN/genética , Girasa de ADN/metabolismo , ADN Superhelicoidal/metabolismo , Diterpenos/química , Diterpenos/toxicidad , Inhibidores Enzimáticos/química , Datos de Secuencia Molecular , Mutagénesis , Análisis de Secuencia de ADN , Streptococcus pneumoniae/enzimología
5.
Methods Mol Biol ; 314: 397-415, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16673896

RESUMEN

Helicases are ubiquitous enzymes that disrupt complementary strands of duplex nucleic acid in a reaction dependent on nucleoside-5'-triphosphate hydrolysis. Helicases are implicated in the metabolism of DNA structures that are generated during replication, recombination, and DNA repair. Furthermore, an increasing number of helicases have been linked to genomic instability and human disease. With the growing interest in helicase mechanism and function, we have set out to describe some basic protocols for biochemical characterization of DNA helicases. Protocols for measuring ATP hydrolysis, DNA binding, and catalytic unwinding activity of DNA helicases are provided. Application of these procedures should enable the researcher to address fundamental questions regarding the biochemical properties of a given helicase, which would serve as a platform for further investigation of its molecular and cellular functions.


Asunto(s)
ADN Helicasas/análisis , ADN Helicasas/metabolismo , ADN Superhelicoidal/metabolismo , Radioquímica/métodos , Adenosina Trifosfatasas/análisis , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Colodión/química , ADN/química , ADN/metabolismo , ADN Helicasas/química , ADN Cruciforme/química , ADN Cruciforme/metabolismo , ADN Superhelicoidal/química , Ensayo de Cambio de Movilidad Electroforética , Humanos , Hidrólisis , Filtros Microporos , Unión Proteica
6.
FEBS J ; 272(24): 6336-43, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16336270

RESUMEN

A catalytic turnover of supercoiled DNA (scDNA) transformation mediated by topoisomerases leads to changes in the linking number (Lk) of the polymeric substrate by 1 or 2 per cycle. As a substrate of the topoisomerization reaction it is chemically identical to its product; even a single catalytic event results in the quantum leap in the scDNA topology. Non-intrusive continuous assay to measure the kinetics of the scDNA topoisomerization was performed. The development of such a technique was hindered because of multiple DNA species of intermediate topology present in the reaction mixture. The interrelation of DNA topology, its hydrodynamics, and optical anisotropy enable us to use the flow linear dichroism technique (FLD) for continuous monitoring of the scDNA topoisomerization reaction. This approach permits us to study the kinetics of DNA transformation catalyzed by eukaryotic topoisomerases I and II, as well as mechanistic characteristics of these enzymes and their interactions with anticancer drugs. Moreover, FLD assay can be applied to any enzymatic reaction that involves scDNA as a substrate. It also provides a new way of screening drugs dynamically and is likely to be potent in various biomedical applications.


Asunto(s)
ADN-Topoisomerasas/metabolismo , ADN Superhelicoidal/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Antineoplásicos/farmacología , Catálisis , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Cinética , Análisis Espectral
7.
Nucleic Acids Res ; 32(19): e153, 2004 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-15520462

RESUMEN

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson-Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37 degrees C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.


Asunto(s)
Cerio/metabolismo , ADN/metabolismo , Ácido Edético/análogos & derivados , Ácido Edético/metabolismo , Ácidos Nucleicos de Péptidos/química , Secuencia de Bases , ADN/química , ADN Ligasas/metabolismo , ADN Superhelicoidal/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Hidrólisis , Datos de Secuencia Molecular , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Especificidad por Sustrato
8.
Acta Biochim Biophys Sin (Shanghai) ; 36(9): 609-17, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15346198

RESUMEN

A eukaryotic cambialistic superoxide dismutase (SOD) has been purified to homogeneity from mature seeds of the disease- and insect-resistant camphor tree (Cinnamomum camphora). Besides the known role of this SOD in protecting cells against oxidative stress, it can induce the cleavage of supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave linear DNA or RNA, demonstrating there is no DNase or RNase in the purified cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. This result indicates that the DNA-cleaving activity requires substrates being topologically constrained. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA), also inhibits the supercoiled DNA-cleaving activity. These results show that the dismutation activity is crucial for the supercoiled DNA cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) shows that these two activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA-cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA are random.


Asunto(s)
Cinnamomum camphora/enzimología , ADN Superhelicoidal/metabolismo , Células Eucariotas/enzimología , Superóxido Dismutasa/farmacología , Catalasa/farmacología , ADN Superhelicoidal/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Etanol/farmacología , Radical Hidroxilo/química , Quelantes del Hierro/farmacología , Manitol/farmacología , Estructura Molecular , Ácido Pentético/farmacología , Extractos Vegetales/química , Especies Reactivas de Oxígeno/metabolismo , Semillas/enzimología , Azida Sódica/farmacología , Relación Estructura-Actividad , Especificidad por Sustrato , Superóxido Dismutasa/química , Superóxido Dismutasa/aislamiento & purificación
9.
J Ethnopharmacol ; 88(2-3): 125-30, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12963131

RESUMEN

Methanolic extracts from seven Plantago species used in traditional medicine for the treatment of cancer, were evaluated for cytotoxic activity against three human cancer cell lines recommended by the National Cancer Institute (NCI, USA). The results showed that Plantago species exhibited cytotoxic activity, showing a certain degree of selectivity against the tested cells in culture. Since the flavonoids are able to strongly inhibit the proliferation of human cancer cell lines, we have identified luteolin-7-O-beta-glucoside as major flavonoid present in most of the Plantago species. Also, we have evaluated this compound and its aglycon, luteolin, for their cytotoxic and DNA topoisomerase I poisons activities. These results could justify the traditional use of the Plantago species and topoisomerase-mediated DNA damage might be a possible mechanism by which flavonoids of Plantago exert their cytotoxicity potential.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Plantago/química , Supervivencia Celular/efectos de los fármacos , Daño del ADN , ADN Superhelicoidal/efectos de los fármacos , ADN Superhelicoidal/metabolismo , Etopósido/farmacología , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Glucósidos/aislamiento & purificación , Glucósidos/farmacología , Humanos , Concentración 50 Inhibidora , Luteolina , Extractos Vegetales/farmacología , Hojas de la Planta/química , Inhibidores de Topoisomerasa I , Células Tumorales Cultivadas
10.
Curr Biol ; 10(14): R526-8, 2000 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-10898993

RESUMEN

The action of individual type II DNA topoisomerases has been followed in real time by observing the elastic response of single DNA molecules to sequential strand passage events. Micromanipulation methods provide a complementary approach to biochemical studies for investigating the mechanism of DNA topoisomerases.


Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , ADN Superhelicoidal/metabolismo , Adenosina Trifosfato/metabolismo , Animales , ADN Superhelicoidal/química , Técnicas In Vitro , Modelos Biológicos , Conformación de Ácido Nucleico
11.
Biochemistry ; 37(46): 16316-24, 1998 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-9819224

RESUMEN

Topoisomerase II is the cytotoxic target for a number of clinically relevant antitumor drugs. Berberrubine, a protoberberine alkaloid which exhibits antitumor activity in animal models, has been identified as a specific poison of topoisomerase II in vitro. Topoisomerase II-mediated DNA cleavage assays showed that berberrubine poisons the enzyme by stabilizing topoisomerase II-DNA cleavable complexes. Subsequent proteinase K treatments revealed that berberrubine-induced DNA cleavage was generated solely by topoisomerase II. Topoisomerase II-mediated DNA religation with elevated temperature revealed a substantial reduction in DNA cleavage induced by berberrubine, to the extent comparable to that of other prototypical topoisomerase II poison, etoposide, suggesting that DNA cleavage involves stabilization of the reversible enzyme-DNA cleavable complex. However, the step at which berberrubine induces cleavable complex may differ from that of etoposide as revealed by the difference in the formation of the intermediate product, nicked DNA. This suggests that berberrubine's primary mode of linear formation may involve trapping nicked molecules, formed at transition from linear to covalently closed circular DNA. Unwinding of the duplex DNA by berberrubine is consistent with an intercalative binding mode for this compound. In addition to the ability to induce the cleavable complex mediated with topoisomerase II, berberrubine at high concentrations was shown to specifically inhibit topoisomerase II catalytic activity. Berberrubine, however, did not inhibit topoisomerase I at concentrations up to 240 microM. Cleavage sites induced by topoisomerase II in the presence of berberrubine and etoposide were mapped in DNA. Berberrubine induces DNA cleavage in a site-specific and concentration-dependent manner. Comparison of the cleavage pattern of berberrubine with that of etoposide revealed that they share many common sites of cleavage. Taken together, these results indicate that berberrubine represents a new class of antitumor agent which exhibits the topoisomerase II poison activity as well as catalytic inhibition activity and may have a potential clinical value in cancer treatment.


Asunto(s)
Alcaloides de Berberina/farmacología , Berberina/análogos & derivados , Berberina/farmacología , Daño del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Catálisis/efectos de los fármacos , ADN Superhelicoidal/efectos de los fármacos , ADN Superhelicoidal/metabolismo , Activación Enzimática/efectos de los fármacos , Etopósido/farmacología , Humanos , Sustancias Intercalantes/farmacología , Mapeo Peptídico , Plantas Medicinales/química , Inhibidores de Topoisomerasa II
12.
Plant Mol Biol ; 37(5): 773-84, 1998 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9678572

RESUMEN

We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial cDNA clones were isolated which were truncated at the 5' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topoisomerase purified to homogeneity. The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg2+. In the presence of Mg2+ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei.


Asunto(s)
ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Genes de Plantas/genética , Pisum sativum/enzimología , Secuencia de Aminoácidos , Cationes Bivalentes , Núcleo Celular/enzimología , Clonación Molecular , ADN-Topoisomerasas de Tipo I/aislamiento & purificación , ADN Complementario/genética , ADN de Plantas/genética , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Expresión Génica , Magnesio , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Pisum sativum/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Análisis de Secuencia de ADN
13.
Proc Natl Acad Sci U S A ; 93(24): 13571-6, 1996 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-8942975

RESUMEN

The Ying-Yang 1 protein (YY1) DNA-binding site functions as an initiator element at which YY1, transcription factor IIB (TFIIB), and RNA polymerase II sponsor basal transcription from a supercoiled DNA template. We show that TFIIB binds to YY1, stabilizing its interaction with DNA, and YY1 contacts the large subunit of polymerase II, directing it to the initiation site. YY1 directs initiation from linear DNA containing mismatched sequences within its binding site, leading us to infer that supercoiling facilitates the separation of DNA strands and to suggest that YY1 likely remains bound to the start site as DNA strands separate during initiation. These results provide a mechanistic basis for transcriptional initiation directed by YY1 in the absence of the TATA box-binding protein.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , ADN/química , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Células HeLa , Humanos , ARN Polimerasa II/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , TATA Box , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIB , Transcripción Genética , Factor de Transcripción YY1
14.
Plant Mol Biol ; 29(4): 703-10, 1995 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-8541497

RESUMEN

A single-strand-specific endonuclease from mung bean sprouts is widely used in molecular biology. However, the biological role of this enzyme is unknown. We studied the spatial and temporal activity of single-stranded DNA endonucleases in mung bean seedling by following enzyme activity that linearizes supercoiled plasmid DNA, a characteristic of this type of enzyme. The formation of a linear molecule from supercoiled DNA was found to occur in two distinguishable steps. The first, which involves introducing a nick into the supercoiled DNA and relaxing it, is very rapid and complete within a few seconds. The second step of cleaving the opposite strand to generate a unit-length linear duplex DNA is a relatively slow process. Analysis of the DNA cleavage sites showed the nuclease preferentially cuts supercoiled DNA at an AT-rich region. Varying levels of nuclease activity could be detected in different tissues of the mung bean seedling. The highest activity was in the root tip and was correlated with histone H1 kinase activity. This implies a link between nuclease activity and cell division. Induction of cell division in mung bean hypocotyls with auxin promoted formation of root primordia and considerably increased the activity of single-stranded DNA endonucleases. The nuclease activity and histone H1 kinase activity were reduced in mung bean cuttings treated with hydroxyurea, but not in cuttings treated with oryzalin. The potential function of single-stranded DNA endonucleases is discussed.


Asunto(s)
División Celular/fisiología , ADN de Plantas/metabolismo , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/metabolismo , Fabaceae/enzimología , Plantas Medicinales , Secuencia de Bases , Clonación Molecular , Reparación del ADN , Replicación del ADN , ADN de Plantas/biosíntesis , ADN Superhelicoidal/metabolismo , Datos de Secuencia Molecular , Protamina Quinasa/análisis , Análisis de Secuencia de ADN , Distribución Tisular
15.
J Med Chem ; 38(6): 1044-7, 1995 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-7699697

RESUMEN

Compounds bearing an acyl group of a various size at 1'-OH of shikonin were synthesized as acyl analogues of shikonin, which was isolated from the root of Lithospermum erythrorhizon, and evaluated for inhibitory effect on topoisomerase-I activity. A selective acylation at 1'-OH of shikonin in the presence of dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine gave rise to a good yield of corresponding acylshikonin derivatives. In general, analogues with an acyl group of shorter chain lengths (C2-C6) exerted a stronger inhibitory action than those with longer chain lengths (C7-C20). While the halogen substitution at C-2 of the acetyl moiety failed to increase the inhibitory potency, the placement of double bonds in the acyl group (C5-C7) augmented the potency remarkably. Of the 32 derivatives evaluated, 15 compounds exhibited a higher inhibitory effect than shikonin. Noteworthy, the inhibitory potency of acetylshikonin, propanoylshikonin, and 4-pentenoylshikonin was approximately 4-fold greater than that of camptothecin. All these data suggest that the size of acyl moiety is important for the enhancement of potency, and the presence of olefinic double bonds is also beneficial.


Asunto(s)
Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/farmacología , Naftoquinonas/síntesis química , Naftoquinonas/farmacología , Inhibidores de Topoisomerasa I , Acetilación , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Superhelicoidal/metabolismo , Células HeLa , Humanos , Extractos Vegetales/farmacología , Raíces de Plantas/química
16.
J Bacteriol ; 177(3): 566-72, 1995 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-7836288

RESUMEN

The recO gene product is required for DNA repair and some types of homologous recombination in wild-type Escherichia coli cells. RecO protein has been previously purified and shown to bind to single- and double-stranded DNA and to promote the renaturation of complementary single-stranded DNA molecules. In this study, purified RecO protein was shown to catalyze the assimilation of single-stranded DNA into homologous superhelical double-stranded DNA, an activity also associated with RecA protein. The RecO protein-promoted strand assimilation reaction requires Mg2+ and is ATP independent. Because of the biochemical similarities between RecO and RecA proteins, the ability of RecO protein to substitute for RecA protein in DNA repair in vivo was also assessed in this study. The results show that overexpression of RecO protein partially suppressed the UV repair deficiency of a recA null mutant and support the hypothesis that RecO and RecA proteins are functionally similar with respect to strand assimilation and the ability to enhance UV survival. These results suggest that RecO and RecA proteins may have common functional properties.


Asunto(s)
Proteínas Bacterianas/fisiología , Reparación del ADN , ADN de Cadena Simple/metabolismo , ADN Superhelicoidal/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Magnesio/farmacología , Mutación , Rayos Ultravioleta
17.
EMBO J ; 13(23): 5764-71, 1994 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-7988572

RESUMEN

In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.


Asunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , Escherichia coli/metabolismo , Rec A Recombinasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Clonación Molecular , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Complementario , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , ADN Superhelicoidal/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Escherichia coli/genética , Biblioteca de Genes , Humanos , Masculino , Recombinasa Rad51 , Rec A Recombinasas/ultraestructura , Testículo/metabolismo
18.
J Biol Chem ; 269(5): 3793-801, 1994 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-8106424

RESUMEN

A 69-kDa protein with topoisomerase I activity has been homogeneously purified from the chloroplasts of pea leaves. The topoisomerase properties are detected in crude lysate of pea chloroplasts using the technique of transferring 32P radioactivity from the 32P-labeled DNA to the protein. The purified enzyme relaxes both positive and negative supercoils in topological steps of unity without requiring magnesium ions. The enzyme is sensitive to topoisomerase I-specific inhibitors like camptothecin and berenil, and unaffected by reagents like novobiocin and doxorubicin at the topoisomerase II-inhibitory dosage. In the presence of the enzyme, supercoiled DNA is nicked, and the 3'-phosphoryl end of the nick becomes covalently linked with the enzyme. A tyrosine residue of the enzyme is responsible for the covalent linkage. Rabbit antiserum raised against the 16-mer peptide spanning the active residues of human topoisomerase I recognizes the 69-kDa protein within the crude lysate of pea chloroplasts as does the antiserum to the purified 69-kDa protein. From the enzymatic characteristics, the protein has been classified as a eukaryotic type I topoisomerase.


Asunto(s)
Cloroplastos/enzimología , ADN-Topoisomerasas de Tipo I/aislamiento & purificación , ADN-Topoisomerasas de Tipo I/metabolismo , Fabaceae/enzimología , Plantas Medicinales , Secuencia de Aminoácidos , Centrifugación por Gradiente de Densidad , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Cromatografía por Intercambio Iónico , ADN-Topoisomerasas de Tipo I/química , ADN Superhelicoidal/metabolismo , Cinética , Datos de Secuencia Molecular , Peso Molecular , Radioisótopos de Fósforo , Especificidad por Sustrato
19.
Nucleic Acids Res ; 20(14): 3679-84, 1992 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-1641333

RESUMEN

Homologous pairing is a key step in homologous genetic recombination. In the early stage of trials for the identification of homologous pairing-promoting proteins from a fission yeast, Schizosaccharomyces pombe, we treated DNA products with phenol in the presence of a salt for the removal of tightly bound proteins from DNA before the assay, but we found that this treatment caused very efficient protein-independent double-strand formation from complementary single-stranded DNAs. Using an assay including the phenol treatment, we detected another species of apparent homologous pairing-promoting proteins in the nuclei, in addition to a homologous pairing-promoting protein consisting of three components which we reported previously. However, studies involving the use of an assay without the phenol-treatments revealed that the second one was not really a homologous pairing-protein. Thus, the protein-independent double-strand formation by phenol-treatment in the presence of a salt could cause the erroneous identification of homologous pairing-promoting proteins.


Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Rec A Recombinasas/metabolismo , Homología de Secuencia de Ácido Nucleico , Bacteriófagos/genética , Composición de Base/genética , Cromatografía , ADN Circular/metabolismo , ADN Superhelicoidal/metabolismo , ADN Viral/metabolismo , Fenol , Fenoles , Recombinación Genética/genética , Schizosaccharomyces/metabolismo
20.
J Nat Prod ; 55(4): 401-13, 1992 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-1324981

RESUMEN

While the design of molecules that inhibit or antagonize the functions of specific macromolecules is now well precedented, in many cases the structural information requisite to the design process is lacking. The tools of molecular biology can now furnish the target macromolecules for use in mechanism-based exploration; highly defined assays can be devised based upon the known biochemistry of these macromolecules to permit the discovery of novel inhibitors or antagonists present in chemical collections. Presently, we describe a set of assays directed toward the discovery of novel inhibitors of eukaryotic topoisomerase I, an enzyme critical to maintenance of chromosomal DNA topology and therefore essential for normal replication and transcription. The identification of chebulagic acid as an extraordinarily potent and mechanically novel inhibitor of topoisomerase I illustrates the potential of this approach.


Asunto(s)
Benzopiranos/farmacología , Glucósidos/farmacología , Extractos Vegetales/farmacología , Inhibidores de Topoisomerasa I , Animales , Benzopiranos/química , Camptotecina/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Evaluación Preclínica de Medicamentos , Electroforesis en Gel de Agar , Glucósidos/química , Taninos Hidrolizables , Espectroscopía de Resonancia Magnética , Conformación de Ácido Nucleico
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