RESUMEN
Bacterial DNA topoisomerase I (topoI) carries out relaxation of negatively supercoiled DNA through a series of orchestrated steps, DNA binding, cleavage, strand passage and religation. The N-terminal domain (NTD) of the type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn(2+) finger motifs in the CTD. The Zn(2+) finger motifs were found to be essential in Escherichia coli topoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial topoI lacks Zn(2+) fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step. Although the NTD binds to DNA in a site-specific fashion to carry out DNA cleavage and religation, the basic residues in CTD bind to non-scissile DNA in a sequence-independent manner to promote the crucial strand passage step during DNA relaxation. The loss of Zn(2+) fingers from the mycobacterial topoI could be associated with Zn(2+) export and homeostasis.
Asunto(s)
Aminoácidos Básicos/metabolismo , Proteínas Bacterianas/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Bacteriano/genética , Mycobacterium smegmatis/enzimología , Aminoácidos Básicos/genética , Proteínas Bacterianas/genética , Sitios de Unión , Transporte Biológico , División del ADN , ADN-Topoisomerasas de Tipo I/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutagénesis Sitio-Dirigida , Mycobacterium smegmatis/genética , Plásmidos/genética , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Dedos de ZincRESUMEN
Bacterial DNA topoisomerase I (topoI) catalyzes relaxation of negatively supercoiled DNA. The enzyme alters DNA topology through protein-operated DNA gate, switching between open and closed conformations during its reaction. We describe the mechanism of inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis topoI by monoclonal antibodies (mAbs) that bind with high affinity and inhibit at 10-50 nM concentration. Unlike other inhibitors of topoisomerases, the mAbs inhibited several steps of relaxation reaction, namely DNA binding, cleavage, strand passage, and enzyme-DNA dissociation. The enhanced religation of the cleaved DNA in presence of the mAb indicated closing of the enzyme DNA gate. The formation of enzyme-DNA heterocatenane in the presence of the mAbs as a result of closing the gate could be inferred by the salt resistance of the complex, visualized by atomic force microscopy and confirmed by fluorescence measurements. Locking the enzyme-DNA complex as a closed clamp restricted the movements of the DNA gate, affecting all of the major steps of the relaxation reaction. Enzyme trapped on DNA in closed clamp conformation formed roadblock for the elongating DNA polymerase. The unusual multistep inhibition of mycobacterial topoisomerases may facilitate lead molecule development, and the mAbs would also serve as valuable tools to probe the enzyme mechanism.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Proteínas Bacterianas/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Inhibidores de Topoisomerasa/farmacología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis/efectos de los fármacos , ADN/química , ADN/genética , ADN/metabolismo , División del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/inmunología , Modelos Biológicos , Modelos Moleculares , Mutación , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , Conformación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Inhibidores de Topoisomerasa/química , Inhibidores de Topoisomerasa/inmunologíaRESUMEN
Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg(2+) dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg(2+) co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg(2+) for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Mycobacterium tuberculosis/enzimología , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Magnesio/metabolismo , Mutagénesis Sitio-Dirigida , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/genética , Especificidad de la Especie , Inhibidores de Topoisomerasa I/farmacologíaRESUMEN
Type IA DNA topoisomerases, typically found in bacteria, are essential enzymes that catalyse the DNA relaxation of negative supercoils. DNA gyrase is the only type II topoisomerase that can carry out the opposite reaction (i.e. the introduction of the DNA supercoils). A number of diverse molecules target DNA gyrase. However, inhibitors that arrest the activity of bacterial topoisomerase I at low concentrations remain to be identified. Towards this end, as a proof of principle, monoclonal antibodies that inhibit Mycobacterium smegmatis topoisomerase I have been characterized and the specific inhibition of Mycobacterium smegmatis topoisomerase I by a monoclonal antibody, 2F3G4, at a nanomolar concentration is described. The enzyme-bound monoclonal antibody stimulated the first transesterification reaction leading to enhanced DNA cleavage, without significantly altering the religation activity of the enzyme. The stimulated DNA cleavage resulted in perturbation of the cleavage-religation equilibrium, increasing single-strand nicks and protein-DNA covalent adducts. Monoclonal antibodies with such a mechanism of inhibition can serve as invaluable tools for probing the structure and mechanism of the enzyme, as well as in the design of novel inhibitors that arrest enzyme activity.
Asunto(s)
Anticuerpos Monoclonales/farmacología , División del ADN , ADN-Topoisomerasas de Tipo I/química , ADN Bacteriano/química , Mycobacterium smegmatis/enzimología , Aductos de ADN , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/inmunología , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN Superhelicoidal , Cinética , Mutación/genéticaRESUMEN
Antigen specific monoclonal antibodies present in crude hybridoma supernatants are normally screened by ELISA on plates coated with the relevant antigen. Screening for inhibitory monoclonals to enzymes would require the evaluation of purified antibodies or antibody containing supernatants for their inhibition of enzyme activity in a separate assay. However, screening for inhibitory antibodies against DNA transacting enzymes such as topoisomerase I (topo I) cannot be done using hybridoma supernatants due to the presence of nucleases in tissue culture media containing foetal calf serum which degrade the DNA substrates upon addition. We have developed a simple and rapid screening procedure for the identification of clones that secrete inhibitory antibodies against mycobacterial topo I using 96 well ELISA microtiter plates. The principle of the method is the selective capture of monoclonal antibodies from crude hybridoma supernatants by topo I that is tethered to the plate through the use of plate-bound polyclonal anti-topo I antibodies. This step allows the nucleases present in the medium to be washed off leaving the inhibitor bound to the tethered enzyme. The inhibitory activity of the captured antibody is assessed by performing an in situ DNA relaxation assay by the addition of supercoiled DNA substrate directly to the microtiter well followed by the analysis of the reaction products by agarose gel electrophoresis. The validity of this method was confirmed by purification of the identified inhibitory antibody and its evaluation in a DNA relaxation assay. Elimination of all enzyme-inhibitory constituents of the culture medium from the well in which the inhibitory antibody is bound to the tethered enzyme may make this method broadly applicable to enzymes such as DNA gyrases, restriction enzymes and other DNA transaction enzymes. Further, the method is simple and avoids the need of prior antibody purification for testing its inhibitory activity.
Asunto(s)
Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , ADN-Topoisomerasas de Tipo I/química , Mycobacterium/enzimología , Inhibidores de Topoisomerasa I , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas/inmunología , ADN-Topoisomerasas de Tipo I/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Ratones , Ratones Endogámicos BALB C , Mycobacterium/inmunologíaRESUMEN
Mycobacterium smegmatis topoisomerase I (MstopoI) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage in the absence of Mg(2+), but religation needs exogenously added Mg(2+). One molecule of Mg(2+) tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim (topoisomerase-primase) domain in MstopoI comprising the Mg(2+) binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg(2+) in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg(2+) dependent for DNA cleavage unlike MstopoI and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg(2)(+)-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.
Asunto(s)
Secuencia de Aminoácidos , Proteínas Bacterianas , ADN-Topoisomerasas de Tipo I , Magnesio/química , Mycobacterium smegmatis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Humanos , Metales , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación de Ácido Nucleico , Alineación de SecuenciaRESUMEN
Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn2+ fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria. This absence of cross-reactivity from other bacteria indicates that mycobacterial topoisomerase I has diverged from Escherichia coli and other bacteria. We have investigated further the immunological properties of the enzyme by raising a panel of monoclonal antibodies that recognises different antigenically active regions of the enzyme and binds it with widely varied affinity. Inhibition of a C-terminal domain-specific antibody binding by enzyme-specific and non-specific oligonucleotides suggests the possibility of using these monoclonal antibodies to probe the structure, function and in vivo role of the enzyme.