RESUMEN
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 walkers are molecular machines that can move with high precision onthe nanoscale due to their structural and functional programmability. Despite recent advances in the field that allow exploring different energy sources, stimuli, and mechanisms of action for these nanomachines, the continuous operation and reusability of DNA walkers remains challenging because in most cases the steps, once taken by the walker, cannot be taken again. Herein we report the path regeneration of a burnt-bridges DNA catenane walker using RNase A. This walker uses a T7RNA polymerase that produces long RNA transcripts to hybridize to the path and move forward while the RNA remains hybridized to the path and blocks it for an additional walking cycle. We show that RNA degradation triggered by RNase A restores the path and returns the walker to the initial position. RNase inhibition restarts the function of the walker.
Asunto(s)
ADN Encadenado/química , Nanotecnología/métodos , ARN/química , Ribonucleasa Pancreática/química , Bacteriófago T7/enzimología , ADN Encadenado/genética , ARN Polimerasas Dirigidas por ADN/química , Hibridación de Ácido Nucleico , ARN/genética , Proteínas Virales/químicaRESUMEN
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
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.
Asunto(s)
Cromosomas Fúngicos/metabolismo , Replicación del ADN/fisiología , ADN Superhelicoidal/metabolismo , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/genética , Cromátides/metabolismo , Cromosomas Fúngicos/química , Cromosomas Fúngicos/genética , ADN-Topoisomerasas/genética , ADN-Topoisomerasas/metabolismo , ADN Encadenado/química , ADN Encadenado/genética , ADN Encadenado/metabolismo , ADN Superhelicoidal/biosíntesis , ADN Superhelicoidal/química , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Conformación de Ácido Nucleico , Plásmidos/química , Plásmidos/genética , Plásmidos/metabolismo , Rotación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN Encadenado/metabolismo , ADN/metabolismo , Anafase/genética , Anafase/fisiología , División Celular/genética , Línea Celular , Línea Celular Tumoral , Centrómero/genética , Cromátides/genética , Cromátides/metabolismo , ADN/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Encadenado/genética , Humanos , CohesinasRESUMEN
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
At each round of cell division, the DNA must be correctly duplicated and distributed between the two daughter cells to maintain genome identity. In order to achieve proper chromosome replication and segregation, sister chromatids must be recognized as such and kept together until their separation. This process of cohesion is mainly achieved through proteinaceous linkages of cohesin complexes, which are loaded on the sister chromatids as they are generated during S phase. Cohesion between sister chromatids must be fully removed at anaphase to allow chromosome segregation. Other (non-proteinaceous) sources of cohesion between sister chromatids consist of DNA linkages or sister chromatid intertwines. DNA linkages are a natural consequence of DNA replication, but must be timely resolved before chromosome segregation to avoid the arising of DNA lesions and genome instability, a hallmark of cancer development. As complete resolution of sister chromatid intertwines only occurs during chromosome segregation, it is not clear whether DNA linkages that persist in mitosis are simply an unwanted leftover or whether they have a functional role. In this review, we provide an overview of DNA linkages between sister chromatids, from their origin to their resolution, and we discuss the consequences of a failure in their detection and processing and speculate on their potential role.
Asunto(s)
Anafase , ADN Encadenado/genética , Inestabilidad Genómica , Animales , Cromátides/química , Cromátides/genética , Segregación Cromosómica , ADN Encadenado/química , HumanosRESUMEN
The capacity of Topoisomerase II (Topo II) to remove DNA catenations that arise after replication is essential to ensure faithful chromosome segregation. Topo II activity is monitored during G2 by a specific checkpoint pathway that delays entry into mitosis until the chromosomes are properly decatenated. Recently, we demonstrated that the mitotic defects that are characteristic of cells depleted of MCPH1 function, a protein mutated in primary microcephaly, are not a consequence of a weakened G2 decatenation checkpoint response. However, the mitotic defects could be accounted for by a minor defect in the activity of Topo II during G2/M. To test this hypothesis, we have tracked at live single cell resolution the dynamics of mitosis in MCPH1 depleted HeLa cells upon catalytic inhibition of Topo II. Our analyses demonstrate that neither chromosome alignment nor segregation are more susceptible to minor perturbation in decatenation in MCPH1 deficient cells, as compared with control cells. Interestingly, MCPH1 depleted cells were more prone to mitotic cell death when decatenation was perturbed. Furthermore, when the G2 arrest that was induced by catalytic inhibition of Topo II was abrogated by Chk1 inhibition, the incidence of mitotic cell death was also increased. Taken together, our data suggest that the MCPH1 lack of function increases mitotic cell hypersensitivity to the catalytic inhibition of Topo II.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas del Citoesqueleto/genética , ADN-Topoisomerasas de Tipo II/genética , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Mitosis/genética , Dominio Catalítico/genética , ADN Encadenado/genética , Inhibidores Enzimáticos/farmacología , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Células HeLa , Humanos , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Two-dimensional agarose gel electrophoresis is the method of choice to identify and quantify all the topological forms DNA molecules can adopt in vivo. Here we describe the materials and protocols needed to analyze catenanes, the natural outcome of DNA replication, in Saccharomyces cerevisiae. We describe the formation of pre-catenanes during replication and how inhibition of topoisomerase 2 leads to the accumulation of intertwined sister duplexes. This knowledge is essential to determine how replication forks blockage or pausing affects the dynamic of DNA topology during replication.
Asunto(s)
Replicación del ADN , ADN Encadenado/genética , Saccharomyces cerevisiae/genética , Replicación del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Encadenado/química , ADN de Hongos/química , ADN de Hongos/genética , Electroforesis en Gel Bidimensional , Conformación de Ácido Nucleico , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
The SgrAI endonuclease displays its maximal activity on DNA with two copies of its recognition sequence, cleaving both sites concertedly. While most restriction enzymes that act concurrently at two sites are tetramers, SgrAI is a dimer in solution. Its reaction at two cognate sites involves the association of two DNA-bound dimers. SgrAI can also bridge cognate and secondary sites, the latter being certain sequences that differ from the cognate by one base-pair. The mechanisms for cognate-cognate and cognate-secondary communications were examined for sites in the following topological relationships: in cis, on plasmids with two sites in a single DNA molecule; on catenanes containing two interlinked rings of DNA with one site in each ring; and in trans, on oligoduplexes carrying either a single site or the DNA termini generated by SgrAI. Both cognate-cognate and cognate-secondary interactions occur through 3-D space and not by 1-D tracking along the DNA. Both sorts of communication arise more readily when the sites are tethered to each other, either in cis on the same molecule of DNA or by the interlinking of catenane rings, than when released from the tether. However, the dimer bound to an oligoduplex carrying either a cognate or a secondary site could be activated to cleave that duplex by interacting with a second dimer bound to the recognition site, provided both duplexes are at least 30 base-pairs long: the second dimer could alternatively be bound to the two duplexes that correspond to the products of DNA cleavage by SgrAI.
Asunto(s)
ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/química , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Secuencia de Bases , Sitios de Unión , ADN/química , ADN/genética , ADN Encadenado/química , ADN Encadenado/genética , ADN Encadenado/metabolismo , Dimerización , Escherichia coli/genética , Conformación de Ácido Nucleico , Plásmidos/química , Plásmidos/genética , Plásmidos/metabolismo , Estructura Cuaternaria de Proteína , Especificidad por SustratoRESUMEN
DNA topoisomerase IIα (TopoIIα) is an essential chromosome-associated enzyme with activity implicated in the resolution of tangled DNA at centromeres before anaphase onset. However, the regulatory mechanism of TopoIIα activity is not understood. Here, we show that PIASy-mediated small ubiquitin-like modifier 2/3 (SUMO2/3) modification of TopoIIα strongly inhibits TopoIIα decatenation activity. Using mass spectrometry and biochemical analysis, we demonstrate that TopoIIα is SUMOylated at lysine 660 (Lys660), a residue located in the DNA gate domain, where both DNA cleavage and religation take place. Remarkably, loss of SUMOylation on Lys660 eliminates SUMOylation-dependent inhibition of TopoIIα, which indicates that Lys660 SUMOylation is critical for PIASy-mediated inhibition of TopoIIα activity. Together, our findings provide evidence for the regulation of TopoIIα activity on mitotic chromosomes by SUMOylation. Therefore, we propose a novel mechanism for regulation of centromeric DNA catenation during mitosis by PIASy-mediated SUMOylation of TopoIIα.
Asunto(s)
Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/genética , ADN-Topoisomerasas de Tipo II/genética , ADN Encadenado/genética , ADN Encadenado/metabolismo , Proteínas de Unión al ADN/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Plk1 (Polo-like kinase 1) has been documented as a critical regulator of many mitotic events. However, increasing evidence supports the notion that Plk1 might also have functions outside of mitosis. Using biochemical fractionation and RNA interference approaches, we found that Plk1 was required for both G(1)/S and G(2)/M phases and that DNA topoisomerase IIalpha (topoIIalpha) was a potential target for Plk1 in both interphase and mitosis. Plk1 phosphorylates Ser(1337) and Ser(1524) of topoIIalpha. Overexpression of an unphosphorylatable topoIIalpha mutant led to S phase arrest, suggesting that Plk1-associated phosphorylation first occurs in S phase. Moreover, overexpression of the unphosphorylatable topoIIalpha mutant activated the ATM/R-dependent DNA damage checkpoint, probably due to reduced catalytic activity of topoIIalpha, and resulted in accumulation of catenated DNA. Finally, we showed that wild type topoIIalpha, but not the unphosphorylatable mutant, was able to rescue topoIIalpha depletion-induced defects in sister chromatid segregation, indicating that Plk1-associated phosphorylation is essential for the functions of topoIIalpha in mitosis.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Cromátides/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Encadenado/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Sustitución de Aminoácidos , Antígenos de Neoplasias/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Cromátides/genética , Daño del ADN/fisiología , ADN-Topoisomerasas de Tipo II/genética , ADN Encadenado/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Quinasa Tipo Polo 1RESUMEN
A challenge for chromosome segregation in all domains of life is the formation of catenated progeny chromosomes, which arise during replication as a consequence of the interwound strands of the DNA double helix. Topoisomerases play a key role in DNA unlinking both during and at the completion of replication. Here we report that chromosome unlinking can instead be accomplished by multiple rounds of site-specific recombination. We show that step-wise, site-specific recombination by XerCD-dif or Cre-loxP can unlink bacterial chromosomes in vivo, in reactions that require KOPS-guided DNA translocation by FtsK. Furthermore, we show that overexpression of a cytoplasmic FtsK derivative is sufficient to allow chromosome unlinking by XerCD-dif recombination when either subunit of TopoIV is inactivated. We conclude that FtsK acts in vivo to simplify chromosomal topology as Xer recombination interconverts monomeric and dimeric chromosomes.