21 research outputs found
The GoldenBricks assembly: A standardized one-shot cloning technique for complete cassette assembly
BBF RFC 92 proposes a new standard assembly method for the Parts Registry. The method makes one-shot cloning of a complete eukaryotic or prokaryotic cassette possible in one day while keeping compatibility with the BBF RFC 10 BioBrick assembly standard
Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes
Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies
Distribution des sites de liaison et activitĂ© de la TopoisomĂ©rase IV sur le gĂ©nome dâEscherichia coli
Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle.Des liens de catĂ©nation sont progressivement crĂ©es lors de la rĂ©plication de lâADN et sont responsables de la cohĂ©sion des chromatides sĆurs. La topoisomĂ©rase IV est une topoisomĂ©rase de type II impliquĂ©e dans la rĂ©solution de ces liens de catĂ©nation accumulĂ©s derriĂšre la fourche de rĂ©plication, et lors de la derniĂšre Ă©tape de sĂ©paration des chromatides sĆurs Ă la fin de la rĂ©plication. Nous avons Ă©tudiĂ© la liaison de la topoIV Ă lâADN ainsi que son activitĂ© catalytique Ă lâaide de mĂ©thodes de biologie molĂ©culaire et de gĂ©nomique. Une expĂ©rience de ChIPseq a rĂ©vĂ©lĂ© que lâinteraction de la topoIV de chez E.coli avec lâADN est contrĂŽlĂ©e par la rĂ©plication. Durant la rĂ©plication, la topoIV a accĂšs Ă des centaines de sites sur lâADN mais ne se lie quâĂ quelques sites oĂč elle exerce son activitĂ© catalytique. La conformation locale de la chromatine et lâexpression des gĂšnes influencent la sĂ©lection de certains sites. De plus, une forte liaison et une activitĂ© catalytique renforcĂ©e a Ă©tĂ© trouvĂ©e au site de rĂ©solution des dimers, dif. Le site dif est situĂ© Ă lâopposĂ© de lâorigine de rĂ©plication dans le macrodomaine ter. Nous avons montrĂ© quâil existe une interaction physique et fonctionnelle entre la topoIV et la recombinase XerCD, qui agit au site dif. Cette interaction est mĂ©diĂ©e par MatP, une protĂ©ine essentielle dans lâorganisation du macrodomaine ter. Lâensemble de ces rĂ©sultats montre que la topoIV, XerCD/dif et MatP Ćuvrent ensemble pour permettre lâĂ©tape finale de sĂ©grĂ©gation des chromosomes lors du cycle cellulaire
Distribution des sites de liaison et activitĂ© de la TopoisomĂ©rase IV sur le gĂ©nome dâEscherichia coli
Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle.Des liens de catĂ©nation sont progressivement crĂ©es lors de la rĂ©plication de lâADN et sont responsables de la cohĂ©sion des chromatides sĆurs. La topoisomĂ©rase IV est une topoisomĂ©rase de type II impliquĂ©e dans la rĂ©solution de ces liens de catĂ©nation accumulĂ©s derriĂšre la fourche de rĂ©plication, et lors de la derniĂšre Ă©tape de sĂ©paration des chromatides sĆurs Ă la fin de la rĂ©plication. Nous avons Ă©tudiĂ© la liaison de la topoIV Ă lâADN ainsi que son activitĂ© catalytique Ă lâaide de mĂ©thodes de biologie molĂ©culaire et de gĂ©nomique. Une expĂ©rience de ChIPseq a rĂ©vĂ©lĂ© que lâinteraction de la topoIV de chez E.coli avec lâADN est contrĂŽlĂ©e par la rĂ©plication. Durant la rĂ©plication, la topoIV a accĂšs Ă des centaines de sites sur lâADN mais ne se lie quâĂ quelques sites oĂč elle exerce son activitĂ© catalytique. La conformation locale de la chromatine et lâexpression des gĂšnes influencent la sĂ©lection de certains sites. De plus, une forte liaison et une activitĂ© catalytique renforcĂ©e a Ă©tĂ© trouvĂ©e au site de rĂ©solution des dimers, dif. Le site dif est situĂ© Ă lâopposĂ© de lâorigine de rĂ©plication dans le macrodomaine ter. Nous avons montrĂ© quâil existe une interaction physique et fonctionnelle entre la topoIV et la recombinase XerCD, qui agit au site dif. Cette interaction est mĂ©diĂ©e par MatP, une protĂ©ine essentielle dans lâorganisation du macrodomaine ter. Lâensemble de ces rĂ©sultats montre que la topoIV, XerCD/dif et MatP Ćuvrent ensemble pour permettre lâĂ©tape finale de sĂ©grĂ©gation des chromosomes lors du cycle cellulaire
Genomic contacts reveal the control of sister chromosome decatenation in E. coli
Abstract In bacteria, chromosome segregation occurs progressively, from the origin to the terminus, a few minutes after the replication of each locus. In-between replication and segregation, sister loci are maintained in an apparent cohesive state by topological links. Whereas topoisomerase IV (Topo IV), the main bacteria decatenase, controls segregation, little is known regarding the influence of the cohesion step on chromosome folding. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, a massive chromosome reorganization takes place, associated with increases in trans-contacts between catenated sister chromatids and in long-range cis-contacts between the terminus and distant loci on the genome. A genetic analysis of these signals allowed us to decipher specific roles for Topo IV and Topo III, an accessory decatenase. Moreover we revealed the role of MatP, the terminus macrodomain organizing system and MukB, the E. coli SMC in organizing sister chromatids tied by persistent catenation links. We propose that large-scale conformation changes observed in these conditions reveal a defective decatenation hub located in the terminus area. Altogether, our findings support a model of spatial and temporal partition of the tasks required for sister chromosome segregation
Note
Bahgat Ali, Farnall Harry, Lacau Pierre, Hassan Hafez, Sayyed Ahmad el-, Haswell C. J. R. Note. In: Comité de Conservation des Monuments de l'Art Arabe. Fascicule 33, exercice 1920-1924, 1928. pp. 270-271
Extended sister-chromosome catenation leads to massive reorganization of the E. coli genome
International audienceAbstract In bacteria, chromosome segregation occurs progressively from the origin to terminus within minutes of replication of each locus. Between replication and segregation, sister loci are held in an apparent cohesive state by topological links. The decatenation activity of topoisomerase IV (Topo IV) is required for segregation of replicated loci, yet little is known about the structuring of the chromosome maintained in a cohesive state. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, massive chromosome reorganization occurs, associated with increased in contacts between nearby loci, likely trans-contacts between sister chromatids, and in long-range contacts between the terminus and distant loci. We deciphered the respective roles of Topo III, MatP and MukB when TopoIV activity becomes limiting. Topo III reduces short-range inter-sister contacts suggesting its activity near replication forks. MatP, the terminus macrodomain organizing system, and MukB, the Escherichia coli SMC, promote long-range contacts with the terminus. We propose that the large-scale conformational changes observed under these conditions reveal defective decatenation attempts involving the terminus area. Our results support a model of spatial and temporal partitioning of the tasks required for sister chromosome segregation
Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome
International audienceCatenation links between sister chromatids are formed progressively during DNA replica-tion and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chro-matin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle
Note à Messieurs les membres du Comité de l'art arabe
Daressy Georges, Farnall Harry, Firth Cecil Mallaby, Darke F. R. H., Haswell C. J. R., Bahgat Ali, Lacau Pierre, Hassan Hafez, Sayyed Ahmad el-. Note à Messieurs les membres du Comité de l'art arabe. In: Comité de Conservation des Monuments de l'Art Arabe. Fascicule 33, exercice 1920-1924, 1928. pp. 268-269