RESUMEN
DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo VI has been proposed to be a DNA decatenase; however, robust evidence and a mechanism for its preferential decatenation activity was lacking. Using single-molecule magnetic tweezers measurements and supporting ensemble biochemistry, we demonstrate that Methanosarcina mazei topo VI preferentially unlinks, or decatenates DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries. In addition, topo VI demonstrates a significant increase in ATPase activity, DNA binding and rate of strand passage, with increasing DNA writhe, providing further evidence that topo VI is a DNA crossing sensor. Our study strongly suggests that topo VI has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes by sensing and preferentially unlinking DNA crossings with geometries close to 90°.
Asunto(s)
Proteínas Arqueales , ADN-Topoisomerasas de Tipo II , ADN Encadenado , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Encadenado/química , ADN Encadenado/genética , ADN Encadenado/metabolismo , Methanosarcina/enzimología , Imagen Individual de Molécula , EstereoisomerismoRESUMEN
Decatenation is a crucial in vivo reaction of DNA topoisomerases in DNA replication and is frequently used in in vitro drug screening. Usually this reaction is monitored using kinetoplast DNA as a substrate, although this assay has several limitations. Here we have engineered a substrate for Tn3 resolvase that generates a singly-linked catenane that can readily be purified from the DNA substrate after restriction enzyme digestion and centrifugation. We show that this catenated substrate can be used with high sensitivity in topoisomerase assays and drug-inhibition assays.
Asunto(s)
ADN-Topoisomerasas/metabolismo , ADN Encadenado/metabolismo , Pruebas de Enzimas/métodos , Secuencia de Bases , Recombinación Genética/genética , Especificidad por Sustrato , Resolvasas de Transposones/metabolismoRESUMEN
DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.
Asunto(s)
Proteínas Arqueales/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Encadenado/metabolismo , Sulfolobus solfataricus/enzimología , Proteínas Arqueales/genética , Secuencia de Bases , ADN-Topoisomerasas de Tipo I/genética , ADN de Archaea/química , ADN de Archaea/genética , ADN de Archaea/metabolismo , ADN Encadenado/química , ADN Encadenado/genética , ADN Concatenado/química , ADN Concatenado/genética , ADN Concatenado/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Calor , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Sulfolobus solfataricus/genéticaRESUMEN
DNA nano-structures present appealing new means for monitoring different molecules. Here, we demonstrate the assembly and utilization of a surface-attached double-stranded DNA catenane composed of two intact interlinked DNA nano-circles for specific and sensitive measurements of the life essential topoisomerase II (Topo II) enzyme activity. Topo II activity was detected via the numeric release of DNA nano-circles, which were visualized at the single-molecule level in a fluorescence microscope upon isothermal amplification and fluorescence labeling. The transition of each enzymatic reaction to a micrometer sized labeled product enabled quantitative detection of Topo II activity at the single decatenation event level rendering activity measurements in extracts from as few as five cells possible. Topo II activity is a suggested predictive marker in cancer therapy and, consequently, the described highly sensitive monitoring of Topo II activity may add considerably to the toolbox of individualized medicine where decisions are based on very sparse samples.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , ADN Encadenado/química , ADN Encadenado/metabolismo , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/metabolismo , Secuencia de Bases , ADN-Topoisomerasas de Tipo II/análisis , ADN Encadenado/genética , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Properly condensed chromosomes are necessary for accurate segregation of the sisters after DNA replication. The Escherichia coli condesin is MukB, a structural maintenance of chromosomes (SMC)-like protein, which forms a complex with MukE and the kleisin MukF. MukB is known to be able to mediate knotting of a DNA ring, an intramolecular reaction. In our investigations of how MukB condenses DNA we discovered that it can also mediate catenation of two DNA rings, an intermolecular reaction. This activity of MukB requires DNA binding by the head domains of the protein but does not require either ATP or its partner proteins MukE or MukF. The ability of MukB to mediate DNA catenation underscores its potential for bringing distal regions of a chromosome together.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , ADN Bacteriano/metabolismo , ADN Encadenado/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Represoras/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Encadenado/química , ADN Encadenado/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas Represoras/química , Proteínas Represoras/genéticaRESUMEN
Topoisomerase IIα (TOP2α) is essential for chromosomal condensation and segregation, as well as genomic integrity. Here we report that RNF168, an E3 ligase mutated in the human RIDDLE syndrome, interacts with TOP2α and mediates its ubiquitylation. RNF168 deficiency impairs decatenation activity of TOP2α and promotes mitotic abnormalities and defective chromosomal segregation. Our data also indicate that RNF168 deficiency, including in human breast cancer cell lines, confers resistance to the anti-cancer drug and TOP2 inhibitor etoposide. We also identify USP10 as a deubiquitylase that negatively regulates TOP2α ubiquitylation and restrains its chromatin association. These findings provide a mechanistic link between the RNF168/USP10 axis and TOP2α ubiquitylation and function, and suggest a role for RNF168 in the response to anti-cancer chemotherapeutics that target TOP2.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Segregación Cromosómica/genética , Anomalías Craneofaciales/genética , ADN Encadenado/metabolismo , Resistencia a Antineoplásicos/genética , Etopósido/farmacología , Etopósido/uso terapéutico , Fibroblastos , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Síndromes de Inmunodeficiencia/genética , Discapacidades para el Aprendizaje/genética , Ratones , Mutagénesis Sitio-Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteínas de Unión a Poli-ADP-Ribosa/antagonistas & inhibidores , Enfermedades de Inmunodeficiencia Primaria , Proteómica , ARN Interferente Pequeño/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/uso terapéutico , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
DNA catenanes are assemblies made up of two or more DNA rings linked together through mechanical bonds, and they are desirable for engineering unique nanoscale devices. However, current methods of synthesizing DNA catenanes rely on the formation of strong linking duplexes between component units to enable interlocking and thus do not permit the synthesis of complex single-stranded DNA structures with freely functioning units. We have recently reported DNA sequences that can thread through a DNA circle without the formation of a linking duplex. Here we show that these unique DNA molecules can be further used to make intricate symmetric or asymmetric DNA [3]catenanes, single-stranded DNA assemblies made up of a central mother ring interlocked to two identical or fraternal twin daughter rings, which have never been reported before. These addressable freely functioning interlocked DNA rings should facilitate the design of elaborate nanoscale machines based on DNA.
Asunto(s)
ADN Encadenado/química , Enzimas de Restricción del ADN , ADN Encadenado/síntesis química , ADN Encadenado/metabolismo , Electroforesis en Gel de Poliacrilamida , Nanoestructuras/química , Técnicas de Amplificación de Ácido NucleicoRESUMEN
Catenation is the process by which cyclic strands are combined like the links of a chain, whereas knotting changes the linking properties of a single strand. In the cell, topoisomerases catalyzing strand passage operations enable the knotting and catenation of DNA so that single- or double-stranded segments can be passed through each other. Here, we use a system of closed DNA structures involving a paranemic motif, called PX-DNA, to bind double strands of DNA together. These PX-cohesive closed molecules contain complementary loops whose linking by Escherichia coli topoisomerase 1 (Topo 1) leads to various types of catenated and knotted structures. We were able to obtain specific DNA topological constructs by varying the lengths of the complementary tracts between the complementary loops. The formation of the structures was analyzed by denaturing gel electrophoresis, and the various topologies of the constructs were characterized using the program Knotilus.
Asunto(s)
ADN Encadenado/química , ADN/química , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Encadenado/metabolismo , Escherichia coli/enzimología , Enlace de Hidrógeno , Modelos Moleculares , Conformación de Ácido Nucleico , Desnaturalización de Ácido NucleicoRESUMEN
The construction of DNA nanostructures from branched DNA motifs, or tiles, typically relies on the use of sticky-ended cohesion, owing to the specificity and programmability of DNA sequences. The stability of such constructs when unligated is restricted to a specific range of temperatures, owing to the disruption of base pairing at elevated temperatures. Paranemic (PX) cohesion was developed as an alternative to sticky ends for the cohesion of large topologically closed species that could be purified reliably on denaturing gels. However, PX cohesion is also of limited stability. In this work, we added sticky-ended interactions to PX-cohesive complexes to create interlocked complexes by functionalizing the sticky ends with psoralen, which can form cross-links between the two strands of a double helix. We were able to reinforce the stability of the constructs by creating covalent linkages between the 3'-ends and 5'-ends of the sticky ends; the sticky ends were added to double crossover domains via 3'-3' and 5'-5' linkages. Catenated arrays were obtained either by enzymatic ligation or by UV cross-linking. We have constructed finite-length one-dimensional arrays linked by interlocking loops and have positioned streptavidin-gold particles on these constructs.
Asunto(s)
ADN Encadenado/química , Nanoestructuras/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Emparejamiento Base , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Encadenado/metabolismo , Escherichia coli/enzimología , Oro/química , Modelos Moleculares , Nanoestructuras/ultraestructura , Nanotecnología/métodos , Conformación de Ácido Nucleico , Motivos de NucleótidosRESUMEN
The structural maintenance of chromosome (SMC) protein complexes cohesin and condensin and the Smc5/6 complex (Smc5/6) are crucial for chromosome dynamics and stability. All contain essential ATPase domains, and cohesin and condensin interact with chromosomes through topological entrapment of DNA. However, how Smc5/6 binds DNA and chromosomes has remained largely unknown. Here, we show that purified Smc5/6 binds DNA through a mechanism that requires ATP hydrolysis by the complex and circular DNA to be established. This also promotes topoisomerase 2-dependent catenation of plasmids, suggesting that Smc5/6 interconnects two DNA molecules using ATP-regulated topological entrapment of DNA, similar to cohesin. We also show that a complex containing an Smc6 mutant that is defective in ATP binding fails to interact with DNA and chromosomes and leads to cell death with concomitant accumulation of DNA damage when overexpressed. Taken together, these results indicate that Smc5/6 executes its cellular functions through ATP-regulated intermolecular DNA linking.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN Encadenado/metabolismo , ADN de Hongos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismo , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Due to the helical structure of DNA the process of DNA replication is topologically complex. Freshly replicated DNA molecules are catenated with each other and are frequently knotted. For proper functioning of DNA it is necessary to remove all of these entanglements. This is done by DNA topoisomerases that pass DNA segments through each other. However, it has been a riddle how DNA topoisomerases select the sites of their action. In highly crowded DNA in living cells random passages between contacting segments would only increase the extent of entanglement. Using molecular dynamics simulations we observed that in actively supercoiled DNA molecules the entanglements resulting from DNA knotting or catenation spontaneously approach sites of nicks and gaps in the DNA. Type I topoisomerases, that preferentially act at sites of nick and gaps, are thus naturally provided with DNA-DNA juxtapositions where a passage results in an error-free DNA unknotting or DNA decatenation.
Asunto(s)
ADN Encadenado/química , ADN Superhelicoidal/química , ADN/química , Replicación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Encadenado/metabolismo , ADN Circular/química , ADN Superhelicoidal/metabolismo , Simulación de Dinámica MolecularRESUMEN
Escherichia coli topoisomerases I and III can decatenate double-stranded DNA (dsDNA) molecules containing single-stranded DNA regions or nicks as well as relax negatively supercoiled DNA. Although the proteins share a mechanism of action and have similar structures, they participate in different cellular processes. Whereas topoisomerase III is a more efficient decatenase than topoisomerase I, the opposite is true for DNA relaxation. In order to investigate the differences in the mechanism of these two prototypical type IA topoisomerases, we studied DNA decatenation at the single-molecule level using braids of intact dsDNA and nicked dsDNA with bulges. We found that neither protein decatenates an intact DNA braid. In contrast, both enzymes exhibited robust decatenation activity on DNA braids with a bulge. The experiments reveal that a main difference between the unbraiding mechanisms of these topoisomerases lies in the pauses between decatenation cycles. Shorter pauses for topoisomerase III result in a higher decatenation rate. In addition, topoisomerase III shows a strong dependence on the crossover angle of the DNA strands. These real-time observations reveal the kinetic characteristics of the decatenation mechanism and help explain the differences between their activities.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , ADN Encadenado/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , ADN Encadenado/química , CinéticaRESUMEN
We previously identified salicylate as a novel catalytic inhibitor of human DNA topoisomerase II (topo II; EC 5.99.1.3) that preferentially targets the alpha isoform by interfering with topo II-mediated DNA cleavage. Many pharmaceuticals and compounds found in foods are salicylate-based. We have now investigated whether these are also catalytic inhibitors of topo II and the structural determinants modulating these effects. We have determined that a number of hydroxylated benzoic acids attenuate doxorubicin-induced DNA damage signaling mediated by the ATM protein kinase and inhibit topo II decatenation activity in vitro with varying potencies. Based on the chemical structures of these and other derivatives, we identified unique properties influencing topo II inhibition, including the importance of substitutions at the 2'- and 5'-positions. We extended our findings to a number of salicylate-based pharmaceuticals including sulfasalazine and diflunisal and found that both were effective at attenuating doxorubicin-induced DNA damage signaling, topo II DNA decatenation and they blocked stabilization of doxorubicin-induced topo II cleavable complexes in cells. In a manner similar to salicylate, we determined that these agents inhibit topo II-mediated DNA cleavage. This was accompanied by a concomitant decrease in topo II-mediated ATP-hydrolysis. Taken together, these findings reveal a novel function for the broader class of salicylate-related compounds and highlight the need for additional studies into whether they may impact the efficacy of chemotherapy regimens that include topo II poisons.
Asunto(s)
Antineoplásicos/química , ADN de Neoplasias/química , Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Modelos Moleculares , Proteínas de Neoplasias/antagonistas & inhibidores , Salicilatos/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Antineoplásicos/antagonistas & inhibidores , Antineoplásicos/farmacología , Biocatálisis/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Encadenado/química , ADN Encadenado/metabolismo , ADN de Cinetoplasto/química , ADN de Cinetoplasto/metabolismo , ADN de Neoplasias/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Diflunisal/química , Diflunisal/farmacología , Doxorrubicina/antagonistas & inhibidores , Doxorrubicina/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Hidrólisis/efectos de los fármacos , Células MCF-7 , Conformación Molecular/efectos de los fármacos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Salicilatos/farmacología , Salicilato de Sodio/análogos & derivados , Salicilato de Sodio/química , Salicilato de Sodio/farmacología , Sulfasalazina/química , Sulfasalazina/farmacologíaRESUMEN
The condensin complex is a key determinant of mitotic chromosome architecture. In addition, condensin promotes resolution of sister chromatids during anaphase, a function that is conserved from prokaryotes to human. Anaphase bridges observed in cells lacking condensin are reminiscent of chromosome segregation failure after inactivation of topoisomerase II (topo II), the enzyme that removes catenanes persisting between sister chromatids following DNA replication. Circumstantial evidence has linked condensin to sister chromatid decatenation but, because of the difficulty of observing chromosome catenation, this link has remained indirect. Alternative models for how condensin facilitates chromosome resolution have been put forward. Here, we follow the catenation status of circular minichromosomes of three sizes during the Saccharomyeces cerevisiae cell cycle. Catenanes are produced during DNA replication and are for the most part swiftly resolved during and following S-phase, aided by sister chromatid separation. Complete resolution, however, requires the condensin complex, a dependency that becomes more pronounced with increasing chromosome size. Our results provide evidence that condensin prevents deleterious anaphase bridges during chromosome segregation by promoting sister chromatid decatenation.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Cromátides/enzimología , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Encadenado/metabolismo , Proteínas de Unión al ADN/fisiología , Complejos Multiproteicos/fisiología , Adenosina Trifosfatasas/metabolismo , Ciclo Celular/genética , Cromátides/química , Segregación Cromosómica , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Recent exon-sequencing studies of human tumours have revealed that subunits of BAF (mammalian SWI/SNF) complexes are mutated in more than 20% of all human malignancies, but the mechanisms involved in tumour suppression are unclear. BAF chromatin-remodelling complexes are polymorphic assemblies that use energy provided by ATP hydrolysis to regulate transcription through the control of chromatin structure and the placement of Polycomb repressive complex 2 (PRC2) across the genome. Several proteins dedicated to this multisubunit complex, including BRG1 (also known as SMARCA4) and BAF250a (also known as ARID1A), are mutated at frequencies similar to those of recognized tumour suppressors. In particular, the core ATPase BRG1 is mutated in 5-10% of childhood medulloblastomas and more than 15% of Burkitt's lymphomas. Here we show a previously unknown function of BAF complexes in decatenating newly replicated sister chromatids, a requirement for proper chromosome segregation during mitosis. We find that deletion of Brg1 in mouse cells, as well as the expression of BRG1 point mutants identified in human tumours, leads to anaphase bridge formation (in which sister chromatids are linked by catenated strands of DNA) and a G2/M-phase block characteristic of the decatenation checkpoint. Endogenous BAF complexes interact directly with endogenous topoisomerase IIα (TOP2A) through BAF250a and are required for the binding of TOP2A to approximately 12,000 sites across the genome. Our results demonstrate that TOP2A chromatin binding is dependent on the ATPase activity of BRG1, which is compromised in oncogenic BRG1 mutants. These studies indicate that the ability of TOP2A to prevent DNA entanglement at mitosis requires BAF complexes and suggest that this activity contributes to the role of BAF subunits as tumour suppressors.
Asunto(s)
Antígenos de Neoplasias/metabolismo , ADN Helicasas/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Encadenado/química , ADN Encadenado/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Anafase , Animales , Antígenos de Neoplasias/genética , Puntos de Control del Ciclo Celular , Cromátides/metabolismo , Ensamble y Desensamble de Cromatina , Segregación Cromosómica , ADN Helicasas/deficiencia , ADN Helicasas/genética , Replicación del ADN , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Fibroblastos , Fase G2 , Células HEK293 , Humanos , Meduloblastoma/genética , Ratones , Mitosis , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas de Unión a Poli-ADP-Ribosa , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
Type II topoisomerases are essential enzymes that regulate DNA topology through a strand-passage mechanism. Some type II topoisomerases relax supercoils, unknot and decatenate DNA to below thermodynamic equilibrium. Several models of this non-equilibrium topology simplification phenomenon have been proposed. The kinetic proofreading (KPR) model postulates that strand passage requires a DNA-bound topoisomerase to collide twice in rapid succession with a second DNA segment, implying a quadratic relationship between DNA collision frequency and relaxation rate. To test this model, we used a single-molecule assay to measure the unlinking rate as a function of DNA collision frequency for Escherichia coli topoisomerase IV (topo IV) that displays efficient non-equilibrium topology simplification activity, and for E. coli topoisomerase III (topo III), a type IA topoisomerase that unlinks and unknots DNA to equilibrium levels. Contrary to the predictions of the KPR model, topo IV and topo III unlinking rates were linearly related to the DNA collision frequency. Furthermore, topo III exhibited decatenation activity comparable with that of topo IV, supporting proposed roles for topo III in DNA segregation. This study enables us to rule out the KPR model for non-equilibrium topology simplification. More generally, we establish an experimental approach to systematically control DNA collision frequency.
Asunto(s)
Topoisomerasa de ADN IV/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Encadenado/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Topoisomerasa de ADN IV/química , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/genética , ADN Encadenado/química , Proteínas de Escherichia coli/genéticaRESUMEN
Topoisomerases (topos) maintain DNA topology and influence DNA transaction processes by catalysing relaxation, supercoiling and decatenation reactions. In the cellular milieu, division of labour between different topos ensures topological homeostasis and control of central processes. In Escherichia coli, DNA gyrase is the principal enzyme that carries out negative supercoiling, while topo IV catalyses decatenation, relaxation and unknotting. DNA gyrase apparently has the daunting task of undertaking both the enzyme functions in mycobacteria, where topo IV is absent. We have shown previously that mycobacterial DNA gyrase is an efficient decatenase. Here, we demonstrate that the strong decatenation property of the enzyme is due to its ability to capture two DNA segments in trans. Topo IV, a strong dedicated decatenase of E. coli, also captures two distinct DNA molecules in a similar manner. In contrast, E. coli DNA gyrase, which is a poor decatenase, does not appear to be able to hold two different DNA molecules in a stable complex. The binding of a second DNA molecule to GyrB/ParE is inhibited by ATP and the non-hydrolysable analogue, AMPPNP, and by the substitution of a prominent positively charged residue in the GyrB N-terminal cavity, suggesting that this binding represents a potential T-segment positioned in the cavity. Thus, after the GyrA/ParC mediated initial DNA capture, GyrB/ParE would bind efficiently to a second DNA in trans to form a T-segment prior to nucleotide binding and closure of the gate during decatenation.
Asunto(s)
Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/metabolismo , ADN/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Arginina/química , Topoisomerasa de ADN IV/química , ADN Encadenado/metabolismo , Escherichia coli/enzimología , Mycobacterium smegmatis/enzimología , Unión ProteicaRESUMEN
Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are for assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA, and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay to examine topoisomerase covalent complexes in vivo, and an assay for measuring DNA cleavage in vitro.
Asunto(s)
ADN-Topoisomerasas/metabolismo , ADN Encadenado/efectos de los fármacos , ADN Superhelicoidal/efectos de los fármacos , Pruebas de Enzimas/métodos , Animales , Complejo Antígeno-Anticuerpo/metabolismo , Extractos Celulares , Membrana Celular/inmunología , Células Cultivadas , División del ADN/efectos de los fármacos , ADN-Topoisomerasas/farmacología , ADN Encadenado/metabolismo , ADN Superhelicoidal/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Ratones , Plásmidos/farmacología , Inhibidores de Topoisomerasa/farmacologíaRESUMEN
Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence in situ hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase IIα, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase IIα in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase IIα inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase IIα, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Centrómero/metabolismo , ADN Helicasas/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , RecQ Helicasas/metabolismo , Anafase , Proteínas de Ciclo Celular/metabolismo , Centrómero/química , Centrómero/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , ADN Encadenado/metabolismo , Fase G2 , Células HeLa , Humanos , Mitosis , Mutagénesis Sitio-Dirigida , Prometafase , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , RecQ Helicasas/antagonistas & inhibidores , RecQ Helicasas/genética , CohesinasRESUMEN
DNA decatenation mediated by Topoisomerase II is required to separate the interlinked sister chromatids post-replication. SGS1, a yeast homolog of the human RecQ family of helicases interacts with Topoisomerase II and plays a role in chromosome segregation, but this functional interaction has yet to be identified in higher organisms. Here, we report a physical and functional interaction of Topoisomerase IIα with RECQL5, one of five mammalian RecQ helicases, during DNA replication. Direct interaction of RECQL5 with Topoisomerase IIα stimulates the decatenation activity of Topoisomerase IIα. Consistent with these observations, RECQL5 co-localizes with Topoisomerase IIα during S-phase of the cell cycle. Moreover, cells with stable depletions of RECQL5 display a slow proliferation rate, a G2/M cell cycle arrest and late S-phase cycling defects. Metaphase spreads generated from RECQL5-depleted cells exhibit undercondensed and entangled chromosomes. Further, RECQL5-depleted cells activate a G2/M checkpoint and undergo apoptosis. These phenotypes are similar to those observed when Topoisomerase II catalytic activity is inhibited. These results reveal an important role for RECQL5 in the maintenance of genomic stability and a new insight into the decatenation process.