RESUMEN
A Mg2+-water bridge between the C-3, C-4 diketo moiety of fluoroquinolones and the conserved amino acid residues in the GyrA/ParC subunit is critical for the binding of a fluoroquinolone to a topoisomerase-DNA covalent complex. The fluoroquinolone UING-5-249 (249) can bind to the GyrB subunit through its C7-aminomethylpyrrolidine group. This interaction is responsible for enhanced activities of 249 against the wild type and quinolone-resistant mutant topoisomerases. To further evaluate the effects of the 249-GyrB interaction on fluoroquinolone activity, we examined the activities of decarboxy- and thio-249 against DNA gyrase and conducted docking studies using the structure of a gyrase-ciprofloxacin-DNA ternary complex. We found that the 249-GyrB interaction rescued the activity of thio-249 but not that of decarboxy-249. A C7-group that binds more strongly to the GyrB subunit may allow for modifications at the C-4 position, leading to a novel compound that is active against the wild type and quinolone-resistant pathogens.
Asunto(s)
Ciprofloxacina/metabolismo , Girasa de ADN/metabolismo , ADN Bacteriano/metabolismo , Fluoroquinolonas/metabolismo , Pirrolidinas/química , Staphylococcus aureus/enzimología , Compuestos de Sulfhidrilo/química , Antibacterianos/química , Antibacterianos/metabolismo , Ciprofloxacina/química , Girasa de ADN/química , Girasa de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Descarboxilación , Escherichia coli/metabolismo , Fluoroquinolonas/química , Pruebas de Sensibilidad Microbiana , Subunidades de ProteínaRESUMEN
Fluoroquinolone-class agents selectively target the bacterial type IIA topoisomerases DNA gyrase and topoisomerase IV, with a few exceptions that target eukaryotic type IIA topoisomerases. Fluoroquinolones bind and stabilize type IIA topoisomerase-DNA covalent complexes that contain a double-strand break. This unique mode of action is referred to as 'topoisomerase poisoning'. We discovered that two novel fluoroquinolones having aryl functionality at the N-1 position, UITT-3-217 (217) and UITT-3-227 (227), could inhibit the catalytic activity of human topoisomerase II without stabilizing topoisomerase-DNA complexes, i.e., without poisoning it. Surprisingly, these compounds are more effective in inhibiting the catalytic activities of human and bacterial topoisomerase I. The National Cancer Institute's 60 human tumor cell lines screen revealed significant anti-proliferative activities with 217 and 227 against the majority of 60 cancer cell lines. A proof of concept in vivo efficacy study using an HT-29 xenograft model of human colorectal cancer showed that 217 could inhibit the proliferation of human colorectal cancer cells to a degree comparable to fluorouracil in mice. Although 227 also exhibited anti-proliferative activity, it was not as effective as 217 in this xenograft model. These novel fluoroquinolones may serve as promising lead compounds for the development of new anticancer drugs.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , ADN-Topoisomerasas de Tipo I/química , Fluoroquinolonas/farmacología , Inhibidores de Topoisomerasa I/farmacología , Animales , Antineoplásicos/química , Apoptosis , Proliferación Celular , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Femenino , Fluoroquinolonas/química , Humanos , Ratones , Ratones Desnudos , Inhibidores de Topoisomerasa I/química , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Structural studies of topoisomerase-fluoroquinolone-DNA ternary complexes revealed a cavity between the quinolone N-1 position and the active site tyrosine. Fluoroquinolone derivatives having positively charged or aromatic moieties extended from the N-1 position were designed to probe for binding contacts with the phosphotyrosine residue in ternary complex. While alkylamine, alkylphthalimide, and alkylphenyl groups introduced at the N-1 position afforded derivatives that maintained modest inhibition of the supercoiling activity of DNA gyrase, none retained ability to poison DNA gyrase. Thus, the addition of a large and/or long moiety at the N-1 position disrupts ternary complex formation, and retained ability to inhibit supercoiling is likely through interference with the strand breakage reaction. Two derivatives were found to possess inhibitory effects on the decatenation activity of human topoisomerase II.
Asunto(s)
Girasa de ADN/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Fluoroquinolonas/química , Tirosina/química , Sitios de Unión , Dominio Catalítico , Girasa de ADN/química , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fluoroquinolonas/síntesis química , Fluoroquinolonas/metabolismo , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Relación Estructura-ActividadRESUMEN
Fluoroquinolones form drug-topoisomerase-DNA complexes that rapidly block transcription and replication. Crystallographic and biochemical studies show that quinolone binding involves a water/metal-ion bridge between the quinolone C3-C4 keto-acid and amino acids in helix-4 of the target proteins, GyrA (gyrase) and ParC (topoisomerase IV). A recent cross-linking study revealed a second drug-binding mode in which the other end of the quinolone, the C7 ring system, interacts with GyrA. We report that addition of a dinitrophenyl (DNP) moiety to the C7 end of ciprofloxacin (Cip-DNP) reduced protection due to resistance substitutions in Escherichia coli GyrA helix-4, consistent with the existence of a second drug-binding mode not evident in X-ray structures of drug-topoisomerase-DNA complexes. Several other C7 aryl fluoroquinolones behaved in a similar manner with particular GyrA mutants. Treatment of E. coli cultures with Cip-DNP selectively enriched an uncommon variant, GyrA-A119E, a change that may impede binding of the dinitrophenyl group at or near the GyrA-GyrA interface. Collectively the data support the existence of a secondary quinolone-binding mode in which the quinolone C7 ring system interacts with GyrA; the data also identify C7 aryl derivatives as a new way to obtain fluoroquinolones that overcome existing GyrA-mediated quinolone resistance.
Asunto(s)
Antibacterianos/química , Girasa de ADN/genética , Fluoroquinolonas/química , Inhibidores de Topoisomerasa II/química , Antibacterianos/farmacología , Girasa de ADN/química , Dinitrofenoles/química , Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Fluoroquinolonas/farmacología , Mutación , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
BACKGROUND: Fluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase-DNA covalent complex as a topoisomerase-fluoroquinolone-DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction. METHODS: We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg(2+)-, Mn(2+)-, or Ca(2+)-supported DNA cleavage activity of Escherichia coli Topo IV. RESULTS: In the absence of any drug, 20-30 mM Mg(2+) was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1mM of either Mn(2+) or Ca(2+) was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg(2+) concentrations where Topo IV alone could not efficiently cleave DNA. CONCLUSIONS AND GENERAL SIGNIFICANCE: At low Mg(2+) concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg(2+) binding to metal binding site B through the structural distortion in DNA. As Mg(2+) concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg(2+) at site B or inhibition the binding of Mg(2+) to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg(2+) binding.
Asunto(s)
División del ADN/efectos de los fármacos , Topoisomerasa de ADN IV/metabolismo , Fluoroquinolonas/farmacología , Magnesio/metabolismo , Sitios de Unión , Calcio/farmacología , Catálisis , Magnesio/farmacologíaRESUMEN
DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys(466) gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly(81) and GyrB-Glu(466) residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.
Asunto(s)
Girasa de ADN/metabolismo , ADN Bacteriano/metabolismo , Fluoroquinolonas/metabolismo , Sustancias Macromoleculares/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Ciprofloxacina/química , Ciprofloxacina/metabolismo , Ciprofloxacina/farmacología , Cristalografía por Rayos X , Girasa de ADN/química , Girasa de ADN/genética , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoroquinolonas/química , Fluoroquinolonas/farmacología , Sustancias Macromoleculares/química , Sustancias Macromoleculares/farmacología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Mutación , Mycobacterium smegmatis/efectos de los fármacos , Conformación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de Proteína , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/metabolismo , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
The increasing occurrence of drug-resistant bacterial infections in the clinic has created a need for new antibacterial agents. Natural products have historically been a rich source of both antibiotics and lead compounds for new antibacterial agents. The natural product simocyclinone D8 (SD8) has been reported to inhibit DNA gyrase, a validated antibacterial drug target, by a unique catalytic inhibition mechanism of action. In this work, we have prepared simplified flavone-based analogues inspired by the complex natural product and evaluated their inhibitory activity and mechanism of action. While two of these compounds do inhibit DNA gyrase, they do so by a different mechanism of action than SD8, namely DNA intercalation.
Asunto(s)
Antibacterianos/química , Girasa de ADN/metabolismo , Escherichia coli/enzimología , Flavonas/química , Inhibidores de Topoisomerasa II/química , Antibacterianos/farmacología , Cumarinas/química , Cumarinas/farmacología , Girasa de ADN/química , Escherichia coli/efectos de los fármacos , Infecciones por Escherichia coli/tratamiento farmacológico , Flavonas/farmacología , Glicósidos/química , Glicósidos/farmacología , Humanos , Modelos Moleculares , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Numerous therapeutic applications have been proposed for molecules that bind heparin-binding proteins. Development of such compounds has primarily focused on optimizing the degree and orientation of anionic groups on a scaffold, but utility of these polyanions has been diminished by their typically large size and non-specific interactions with many proteins. In this study N-arylacyl O-sulfonated aminoglycosides were synthesized and evaluated for their ability to selectively inhibit structurally similar bacterial and human topoisomerases. It is demonstrated that the structure of the aminoglycoside and of the N-arylacyl moiety imparts selective inhibition of different topoisomerases and alters mechanism. The results here outline a strategy that will be applicable to identifying small, structurally defined oligosaccharides that bind heparin-binding proteins with a high degree of selectivity.
RESUMEN
During a survey of actinobacteria known to suppress the growth of Streptomyces scabies (the causative agent of potato scab disease) in vivo, six new rhamnosylated alkaloids, the solphenazines A-F (1-6), were isolated from a biological control strain of Streptomyces (DL-93). The known rhamnosyl analogue of paraben (9) was also isolated along with a new rhamnosylated derivative of N-methyl-p-aminobenzoic acid (10). None of the compounds exhibited any antibacterial or antifungal activity against a standard panel of microorganisms, but compounds 1, 2, and 6 displayed some cytotoxicity against HCT-116 cancer cells. Additional in vitro testing provided data suggesting that the cytotoxic activity is not due to DNA intercalation or topoisomerase inhibition.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Glicósidos/aislamiento & purificación , Fenazinas/aislamiento & purificación , Enfermedades de las Plantas/microbiología , Streptomyces , Ácido 4-Aminobenzoico/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Chlorocebus aethiops , Ensayos de Selección de Medicamentos Antitumorales , Glicósidos/química , Glicósidos/farmacología , Células HCT116 , Humanos , Riñón/citología , Riñón/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Fenazinas/química , Fenazinas/farmacología , Streptomyces/química , Streptomyces/efectos de los fármacos , Streptomyces/genética , Streptomyces/crecimiento & desarrolloRESUMEN
Simocyclinone D8 is an antibiotic isolated from Streptomyces antibioticus Tü 6040 that inhibits the supercoiling activity of DNA gyrase. It also exhibits an inhibitory effect on human topoisomerase II and an antiproliferative activity against some cancer cell lines. Our biochemical studies have revealed that simocyclinone D8 can inhibit the catalytic activity of human topoisomerase I. Thus, simocyclinone D8 is a dual catalytic inhibitor of human topoisomerases I and II.
Asunto(s)
Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/farmacología , Cumarinas/química , Cumarinas/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Glicósidos/química , Glicósidos/farmacología , Humanos , Estructura Molecular , Streptomyces antibioticusRESUMEN
Bacterial resistance presents a difficult issue for fluoroquinolone treatment of bacterial infections. In previous work, we reported that 8-methoxy-quinazoline-2,4-diones are active against quinolone-resistant mutants of Escherichia coli. Here, we demonstrate the activity of a representative 8-methoxy-quinazoline-2,4-dione against quinolone-resistant gyrases. Furthermore, 8-methoxy-quinazoline-2,4-dione and other diones are shown to inhibit Staphylococcus aureus gyrase and topoisomerase IV with similar degrees of efficacy, suggesting that the diones might act as dual-targeting agents against S. aureus.
Asunto(s)
Antibacterianos/farmacología , Fluoroquinolonas/farmacología , Antibacterianos/química , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/metabolismo , Activación Enzimática/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Fluoroquinolonas/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Proteínas Virales/metabolismoRESUMEN
INTRODUCTION: Simocyclinone D-8 (SD8), a semi-synthetic compound derived from yeast, has been shown to decrease the proliferation of MCF-7 breast cancer cells. It has been shown to be a potent bacterial DNA gyrase inhibitor, a homologue of human topoisomerase II (hTopoII). We tested SD8 activity alone and in combination with cisplatin against malignant mesothelioma (MM) and non-small cell cancer (NSCLC) cell lines. METHODS: Inhibition of hTopoII supercoiling function by SD8 and a known hTopoII poison, etoposide, were done by in vitro assay using purified hTopoII and kinetoplast DNA as the substrate. The DNA products were analyzed by agarose gel electrophoresis after treatment with increasing concentrations of each drug. Mesothelioma cell lines (H2373, H2461 and H2596) and NSCLC cell lines (H2030, H460, and H2009) grown in RPMI with 10% calf serum were used. Non-malignant mesothelial cells, LP9, were grown in 1:1 ratio of MCDB:199E medium supplemented with 15% calf serum, 0.4 microg/mL hydrocortisone, and 15 ng/mL epidermal grown factor. Cell proliferation assays were performed in 96-well plates using the CCK-8 kit (Dojindo inc.). Cells were treated for 72 h with various SD8 concentrations and controls containing equal volume of the vehicle, DMSO. Treated cells were assayed for the induction of apoptosis with poly ADP-ribose polymerase-1 (PARP) cleavage assay. RESULTS: Biochemical assays revealed that the IC(50) for hTopoII inhibition was 100 microM for SD8 and 400 microM for etoposide. SD8 inhibited hTopoII function without inducing DNA cleavage events. SD8 inhibited the growth of NSCLC and Mesothelioma cells with IC(50) ranging from 75-125 microM. Furthermore, SD8 was not toxic to non-transformed primary mesothelial cell line, LP9 at the IC(50) doses. SD8 induced apoptosis in all cell lines tested. CONCLUSIONS: SD8 inhibits hTopoII in vitro without inducing DNA strands breaks and has significant activity against NSCLC and MM cell lines. While doses required for SD8 anticancer activity are unlikely to be achieved in vivo, chemical modifications to SD8 to increase its potency could lead to improved therapies for these diseases.
Asunto(s)
Biocatálisis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores de Topoisomerasa II , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cumarinas/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Glicósidos/farmacología , HumanosRESUMEN
Simocyclinone D8 (SD8) exhibits antibiotic activity against gram-positive bacteria but not against gram-negative bacteria. The molecular basis of the cytotoxicity of SD8 is not fully understood, although SD8 has been shown to inhibit the supercoiling activity of Escherichia coli gyrase. To understand the mechanism of SD8, we have employed biochemical assays to directly measure the sensitivities of E. coli and Staphylococcus aureus type II topoisomerases to SD8 and microarray analysis to monitor the cellular responses to SD8 treatment. SD8 is a potent inhibitor of either E. coli or S. aureus gyrase. In contrast, SD8 exhibits only a moderate inhibitory effect on S. aureus topoisomerase IV, and E. coli topoisomerase IV is virtually insensitive to SD8. The antimicrobial effect of SD8 against E. coli has become evident in the absence of the AcrB multidrug efflux pump. As expected, SD8 treatment exhibits the signature responses to the loss of supercoiling activity in E. coli: upregulation of gyrase genes and downregulation of the topoisomerase I gene. Unlike quinolone treatment, however, SD8 treatment does not induce the SOS response. These results suggest that DNA gyrase is the target of SD8 in both gram-positive and gram-negative bacteria and that the lack of the antibacterial effect against gram-negative bacteria is due, in part, to the activity of the AcrB efflux pump.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Streptomyces antibioticus/metabolismo , Antraquinonas/química , Antraquinonas/farmacología , Cumarinas/química , Cumarinas/farmacología , Medios de Cultivo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicósidos/química , Glicósidos/farmacología , Pruebas de Sensibilidad Microbiana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrollo , Streptomyces antibioticus/crecimiento & desarrollo , Inhibidores de Topoisomerasa IIRESUMEN
We have identified a small library of novel substituted 9-aminoacridine derivatives that inhibit cell proliferation of pancreatic cancer cell lines by inducing apoptosis [Goodell, J.R. et al., 2008. J. Med. Chem. 51, 179-182.]. To further investigate their antiproliferative activities, we have assessed the antiproliferative activity of these acridine-based compounds against several pancreatic cancer cell lines. All four compounds used in this study inhibited the proliferation of pancreatic cancer cell lines in vitro. In addition, we have employed a xenograft tumor model and found that these compounds also inhibit the proliferation of pancreatic cancer in vivo. In light of the potential importance of the anticancer activity of these acridine-based compounds, we have conducted a series of biochemical assays to determine the effect of these compounds on human topoisomerase II. Unlike amsacrine, these compounds do not poison topoisomerase II. Similar to amsacrine, however, these compounds intercalate into DNA in a way that they would alter the apparent topology of the DNA substrate. Thus, inhibition of the relaxation activity of topoisomerase II by these compounds has been reexamined using a DNA strand passage assay. We have found that these compounds, indeed, inhibit the catalytic activity of topoisomerase II. Thus, these novel acridine-based compounds with anti-pancreatic cancer activity are catalytic inhibitors, not poisons, of human topoisomerase II.
Asunto(s)
Acridinas/química , Antineoplásicos/química , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/enzimología , Inhibidores de Topoisomerasa II , Animales , Antineoplásicos/metabolismo , Biocatálisis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Femenino , Humanos , Sustancias Intercalantes/química , Sustancias Intercalantes/metabolismo , Sustancias Intercalantes/farmacología , Ratones , Neoplasias Pancreáticas/patología , Trasplante HeterólogoRESUMEN
The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created through the insertion and deletion of specific uridylates. The editing reactions are required to be highly specific in order to ensure that functional open reading frames are created in edited mRNAs and that potentially deleterious modification of normally nonedited sequence does not occur. Selection-amplification and mutagenesis were previously used to identify the optimal sequence requirements for in vitro editing. There is, however, a minority of natural editing sites with suboptimal sequence. Several cis-acting elements, obtained from an in vitro selection, are described here that are able to compensate for a suboptimal editing site. An A + U sequence element within the 5'-untranslated region of cytochrome b mRNA from Leishmania tarentolae is also demonstrated to function as a cis-acting guide RNA and is postulated to compensate for a suboptimal editing site in vivo. Two proteins within an enriched editing extract are UV-cross-linked to two different in vitro selected editing substrates more efficiently than poorly edited RNAs. The results suggest that these proteins contribute to the specificity of the editing reaction.
Asunto(s)
Kinetoplastida/genética , Edición de ARN , Secuencias Reguladoras de Ácido Ribonucleico/fisiología , Animales , Secuencia de Bases , ARN , ARN Mitocondrial , ARN Protozoario/genética , Especificidad por Sustrato , Uridina Monofosfato/metabolismoRESUMEN
The coding sequence of several mitochondrial mRNAs of the trypanosomatid family of protozoa is created by the guide RNA-directed insertion and deletion of uridylates (Us). Selection-amplification was used to explore the sequence and structure of the guide RNA and mRNA required for efficient insertional editing within a mitochondrial extract prepared from Leishmania tarentolae. This study identifies several novel features of the editing reaction in addition to several that are consistent with the previous mutagenesis and phylogenetic analysis of the reaction in Trypanosoma brucei, a distantly related trypanosomatid. Specifically, there is a strong bias against cytidines 5' of the editing sites and guanosines immediately 3' of guiding nucleotides. U insertions are directed both 5' and 3' of a genomically encoded U, which was previously assumed not to occur. Base pairing immediately flanking an editing site can significantly stimulate the editing reaction and affect the reaction fidelity but is not essential. Likewise, single-stranded RNA in the region upstream of the editing site, not necessarily immediately adjacent, can facilitate editing but is also not essential. The editing of an RNA containing many of the optimal features is linear with increasing quantities of extract permitting specific activity measurements to be made that are not possible with previously described T. brucei and L. tarentolae assays. The reaction catalyzed by the L. tarentolae extract can be highly accurate, which does not support a proposed model for editing that was based largely on the inaccuracy of an earlier in vitro reaction.
Asunto(s)
Leishmania/genética , Leishmania/fisiología , Edición de ARN/fisiología , ARN Guía de Kinetoplastida/metabolismo , Animales , Emparejamiento Base , ARN Mensajero/metabolismo , Uridina/metabolismoRESUMEN
The coding sequence within several mitochondrial mRNAs of the trypanosomatid protozoa is created through editing by the precise insertion and deletion of U nucleotides. The biochemical characterisation of the editing reaction in the Leishmania genus of the trypanosomatids has been hindered by the lack of a direct in vitro assay. We describe here the first direct assay for the detection of guide RNA-directed editing mediated by a mitochondrial extract prepared from two independent isolates of Leishmania tarentolae. The assay enabled the editing activity within a L. tarentolae mitochondrial extract to be significantly enriched and will facilitate the characterisation of the editing reaction. The results suggest that the difficulty in establishing an assay for the L. tarentolae reaction was not simply a result of the catalytic machinery being limiting but rather reflected the presence of constraints on both the guide RNA and mRNA sequences.