RESUMO
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.
Assuntos
Proteínas Arqueais/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Catenado/metabolismo , Sulfolobus solfataricus/enzimologia , Proteínas Arqueais/genética , Sequência de Bases , DNA Topoisomerases Tipo I/genética , DNA Arqueal/química , DNA Arqueal/genética , DNA Arqueal/metabolismo , DNA Catenado/química , DNA Catenado/genética , DNA Concatenado/química , DNA Concatenado/genética , DNA Concatenado/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Temperatura Alta , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Sulfolobus solfataricus/genéticaRESUMO
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.
Assuntos
Antígenos de Neoplasias/metabolismo , DNA Helicases/metabolismo , DNA Topoisomerases Tipo II/metabolismo , DNA Catenado/química , DNA Catenado/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Anáfase , Animais , Antígenos de Neoplasias/genética , Pontos de Checagem do Ciclo Celular , Cromátides/metabolismo , Montagem e Desmontagem da Cromatina , Segregação de Cromossomos , DNA Helicases/deficiência , DNA Helicases/genética , Replicação do DNA , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Fibroblastos , Fase G2 , Células HEK293 , Humanos , Meduloblastoma/genética , Camundongos , Mitose , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas de Ligação a Poli-ADP-Ribose , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA Catenado/química , DNA Catenado/metabolismo , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/metabolismo , Sequência de Bases , DNA Topoisomerases Tipo II/análise , DNA Catenado/genética , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
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.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA Bacteriano/metabolismo , DNA Catenado/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Catenado/química , DNA Catenado/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas Repressoras/química , Proteínas Repressoras/genéticaRESUMO
During chromosome duplication the parental DNA molecule becomes overwound, or positively supercoiled, in the region ahead of the advancing replication fork. To allow fork progression, this superhelical tension has to be removed by topoisomerases, which operate by introducing transient DNA breaks. Positive supercoiling can also be diminished if the advancing fork rotates along the DNA helix, but then sister chromatid intertwinings form in its wake. Despite these insights it remains largely unknown how replication-induced superhelical stress is dealt with on linear, eukaryotic chromosomes. Here we show that this stress increases with the length of Saccharomyces cerevisiae chromosomes. This highlights the possibility that superhelical tension is handled on a chromosome scale and not only within topologically closed chromosomal domains as the current view predicts. We found that inhibition of type I topoisomerases leads to a late replication delay of longer, but not shorter, chromosomes. This phenotype is also displayed by cells expressing mutated versions of the cohesin- and condensin-related Smc5/6 complex. The frequency of chromosomal association sites of the Smc5/6 complex increases in response to chromosome lengthening, chromosome circularization, or inactivation of topoisomerase 2, all having the potential to increase the number of sister chromatid intertwinings. Furthermore, non-functional Smc6 reduces the accumulation of intertwined sister plasmids after one round of replication in the absence of topoisomerase 2 function. Our results demonstrate that the length of a chromosome influences the need of superhelical tension release in Saccharomyces cerevisiae, and allow us to propose a model where the Smc5/6 complex facilitates fork rotation by sequestering nascent chromatid intertwinings that form behind the replication machinery.
Assuntos
Cromossomos Fúngicos/metabolismo , Replicação do DNA/fisiologia , DNA Super-Helicoidal/metabolismo , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/genética , Cromátides/metabolismo , Cromossomos Fúngicos/química , Cromossomos Fúngicos/genética , DNA Topoisomerases/genética , DNA Topoisomerases/metabolismo , DNA Catenado/química , DNA Catenado/genética , DNA Catenado/metabolismo , DNA Super-Helicoidal/biossíntese , DNA Super-Helicoidal/química , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , Rotação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase II/farmacologiaRESUMO
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.
Assuntos
DNA Catenado/química , DNA Super-Helicoidal/química , DNA/química , Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , DNA Catenado/metabolismo , DNA Circular/química , DNA Super-Helicoidal/metabolismo , Simulação de Dinâmica MolecularRESUMO
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.
Assuntos
DNA Catenado/química , Enzimas de Restrição do DNA , DNA Catenado/síntese química , DNA Catenado/metabolismo , Eletroforese em Gel de Poliacrilamida , Nanoestruturas/química , Técnicas de Amplificação de Ácido NucleicoRESUMO
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.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Catenado/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , DNA Catenado/química , CinéticaRESUMO
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.
Assuntos
Adenosina Trifosfatases/fisiologia , Cromátides/enzimologia , DNA Topoisomerases Tipo II/metabolismo , DNA Catenado/metabolismo , Proteínas de Ligação a DNA/fisiologia , Complexos Multiproteicos/fisiologia , Adenosina Trifosfatases/metabolismo , Ciclo Celular/genética , Cromátides/química , Segregação de Cromossomos , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
DNA Topoisomerase IV/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Catenado/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , DNA Topoisomerase IV/química , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Catenado/química , Proteínas de Escherichia coli/genéticaRESUMO
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.
Assuntos
Antígenos de Neoplasias/metabolismo , Ciclo Celular , DNA Topoisomerases Tipo II/metabolismo , DNA Catenado/metabolismo , Proteínas de Ligação a DNA/metabolismo , RecQ Helicases/metabolismo , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Proliferação de Células , Aberrações Cromossômicas , Exodesoxirribonucleases/metabolismo , Humanos , Metáfase/genética , RecQ Helicases/antagonistas & inibidores , Helicase da Síndrome de WernerRESUMO
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.
Assuntos
DNA Girase/metabolismo , DNA Topoisomerase IV/metabolismo , DNA/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Arginina/química , DNA Topoisomerase IV/química , DNA Catenado/metabolismo , Escherichia coli/enzimologia , Mycobacterium smegmatis/enzimologia , Ligação ProteicaRESUMO
The organisation of mammalian mitochondrial DNA (mtDNA) is more complex than usually assumed. Despite often being depicted as a simple circle, the topology of mtDNA can vary from supercoiled monomeric circles over catenanes and oligomers to complex multimeric networks. Replication of mtDNA is also not clear cut. Two different mechanisms of replication have been found in cultured cells and in most tissues: a strand-asynchronous mode involving temporary RNA coverage of one strand, and a strand-coupled mode rather resembling conventional nuclear DNA replication. In addition, a recombination-initiated replication mechanism is likely to be associated with the multimeric mtDNA networks found in human heart. Although an insight into the general principles and key factors of mtDNA organisation and maintenance has been gained over the last few years, there are many open questions regarding replication initiation, termination and physiological factors determining mtDNA organisation and replication mode. However, common themes in mtDNA maintenance across eukaryotic kingdoms can provide valuable lessons for future work.
Assuntos
Replicação do DNA , DNA Mitocondrial/metabolismo , Animais , DNA Catenado/metabolismo , DNA Mitocondrial/genética , Humanos , Mamíferos , Mitocôndrias Cardíacas/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Recombinação Genética , Origem de ReplicaçãoRESUMO
Sister chromatid cohesion is mediated by DNA catenation and proteinaceous cohesin complexes. The recent visualization of PICH (Plk1-interacting checkpoint helicase)-coated DNA threads in anaphase cells raises new questions as to the role of DNA catenation and its regulation in time and space. In the present study we show that persistent DNA catenation induced by inhibition of Topoisomerase-IIalpha can contribute to sister chromatid cohesion in the absence of cohesin complexes and that resolution of catenation is essential for abscission. Furthermore, we use an in vitro chromatid separation assay to investigate the temporal and functional relationship between cohesin removal and Topoisomerase-IIalpha-mediated decatenation. Our data suggest that centromere decatenation can occur only after separase activation and cohesin removal, providing a plausible explanation for the persistence of centromere threads after anaphase onset.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Catenado/metabolismo , DNA/metabolismo , Anáfase/genética , Anáfase/fisiologia , Divisão Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Centrômero/genética , Cromátides/genética , Cromátides/metabolismo , DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA Catenado/genética , Humanos , CoesinasRESUMO
A novel series of 4ß-[(4-substituted) aroylthiourea] derivatives of podophyllotoxin were synthesized and their abilities to inhibit the growth of cancer cells were investigated by MTT assay. Compound 4a possessed the highest cytotoxicity on HepG2, A549 and HCT-116 cancer cell lines with the IC(50) values of 0.1 µM. Apoptosis in HCT-116 cells induced by compound 4a was observed by Hoechst33342-Propidium iodide (PI) and acridine orange (AO)-ethidium bromide (EB) double staining assays. DNA flow cytometric analysis revealed that 4a induced cell cycle arrest at G2/M phase and kDNA decatenation assay indicated that 4a inhibited topoisomerase IIα-mediated kDNA decatenation. Our results indicated that compound 4a possessed promising antitumor activity, which need to be studied further.
Assuntos
Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Podofilotoxina/síntese química , Podofilotoxina/farmacologia , Tioureia/síntese química , Tioureia/farmacologia , Antígenos de Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , DNA Catenado/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células HCT116 , Células Hep G2 , Humanos , Concentração Inibidora 50 , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Estrutura Molecular , Podofilotoxina/análogos & derivados , Coloração e Rotulagem , Relação Estrutura-Atividade , Tioureia/análogos & derivados , Fatores de TempoRESUMO
The human Ena/Vasp-like (EVL) protein is considered to be a bifunctional protein, involved in both actin remodeling and homologous recombination. In the present study, we found that human EVL forms heat-stable multimers of circular single-stranded DNA (ssDNA) molecules in the presence of a type I topoisomerase in vitro. An electron microscopic analysis revealed that the heat-stable ssDNA multimers formed by EVL and topoisomerase were ssDNA catemers. The ssDNA catenation did not occur when either EVL or topoisomerase was omitted from the reaction mixture. A deletion analysis revealed that the ssDNA catenation completely depended on the annealing activity of EVL. Human EVL was captured from a human cell extract by TOPO IIIα-conjugated beads, and the interaction between EVL and TOPO IIIα was confirmed by a surface plasmon resonance analysis. Purified TOPO IIIα catalyzed the ssDNA catenation with EVL as efficiently as the Escherichia coli topoisomerase I. Since the ssDNA cutting and rejoining reactions, which are the sub-steps of ssDNA catenation, may be an essential process in homologous recombination, EVL and TOPO IIIα may function in the processing of DNA intermediates formed during homologous recombination.
Assuntos
Moléculas de Adesão Celular/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Catenado/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Catenado/ultraestrutura , DNA de Cadeia Simples/ultraestrutura , Humanos , Modelos BiológicosRESUMO
Leishmania actin (LdACT) is an unconventional form of eukaryotic actin in that it markedly differs from other actins in terms of its filament forming as well as toxin and DNase-1-binding properties. Besides being present in the cytoplasm, cortical regions, flagellum and nucleus, it is also present in the kinetoplast where it appears to associate with the kinetoplast DNA (kDNA). However, nothing is known about its role in this organelle. Here, we show that LdACT is indeed associated with the kDNA disc in Leishmania kinetoplast, and under in vitro conditions, it specifically binds DNA primarily through electrostatic interactions involving its unique DNase-1-binding region and the DNA major groove. We further reveal that this protein exhibits DNA-nicking activity which requires its polymeric state as well as ATP hydrolysis and through this activity it converts catenated kDNA minicircles into open form. In addition, we show that LdACT specifically binds bacterial type II topoisomerase and inhibits its decatenation activity. Together, these results strongly indicate that LdACT could play a critical role in kDNA remodeling.
Assuntos
Actinas/metabolismo , DNA Topoisomerases Tipo II/metabolismo , DNA de Cinetoplasto/metabolismo , Leishmania/metabolismo , Actinas/química , Animais , Linhagem Celular , Cromatina , DNA Catenado/metabolismo , Desoxirribonuclease I/metabolismo , Escherichia coli/enzimologia , Leishmania/genéticaRESUMO
We have studied the stimulation of topoisomerase IV (Topo IV) by the C-terminal AAA+ domain of FtsK. These two proteins combine to assure proper chromosome segregation in the cell. Stimulation of Topo IV activity was dependent on the chirality of the DNA substrate: FtsK stimulated decatenation of catenated DNA and relaxation of positively supercoiled [(+)ve sc] DNA, but inhibited relaxation of negatively supercoiled [(-)ve sc] DNA. The DNA translocation activity of FtsK was not required for stimulation, but was required for inhibition. DNA chirality did not affect any of the activities of FtsK, suggesting that FtsK possesses an inherent Topo IV stimulatory activity that is presumably mediated by protein-protein interactions, the stability of Topo IV on the DNA substrate dictated the effect observed. Inhibition occurs because FtsK can strip distributively acting topoisomerase off (-)ve scDNA, but not from either (+)ve scDNA or catenated DNA where the enzyme acts processively. Our analyses suggest that FtsK increases the efficiency of trapping of the transfer segment of DNA during the catalytic cycle of the topoisomerase.
Assuntos
DNA Topoisomerase IV/metabolismo , DNA Catenado/metabolismo , DNA Super-Helicoidal/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , DNA Catenado/química , DNA Super-Helicoidal/química , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Estrutura Terciária de ProteínaRESUMO
Sister chromatid cohesion, crucial for faithful segregation of replicated chromosomes in eukaryotes, is mediated by the multi-subunit protein complex cohesin. The Saccharomyces cerevisiae plasmid 2 micron circle mimics chromosomes in assembling cohesin at its partitioning locus. The plasmid is a multi-copy selfish DNA element that resides in the nucleus and propagates itself stably, presumably with assistance from cohesin. In metaphase cell lysates, or fractions enriched for their cohesed state by sedimentation, plasmid molecules are trapped topologically by the protein ring formed by cohesin. They can be released from cohesin's embrace either by linearizing the DNA or by cleaving a cohesin subunit. Assays using two distinctly tagged cohesin molecules argue against the hand-cuff (an associated pair of monomeric cohesin rings) or the bracelet (a dimeric cohesin ring) model as responsible for establishing plasmid cohesion. Our cumulative results most easily fit a model in which a single monomeric cohesin ring, rather than a series of such rings, conjoins a pair of sister plasmids. These features of plasmid cohesion account for its sister-to-sister mode of segregation by cohesin disassembly during anaphase. The mechanistic similarities of cohesion between mini-chromosome sisters and 2 micron plasmid sisters suggest a potential kinship between the plasmid partitioning locus and centromeres.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Catenado/química , Plasmídeos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , DNA Catenado/metabolismo , DNA Fúngico/química , DNA Fúngico/metabolismo , Imunoprecipitação , Modelos Genéticos , Plasmídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , CoesinasRESUMO
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°.