Characterization of the sister chromatid cohesion step of the E. coli cell cycle – SISTERS

We have developed a genetic tool allowing to measure the distance between sister chromatid. This tool reach a resolution limit that cannot be achieved by conventional fluorescence microscopes. We have coupled our genetic analysis with molecular and cellular assays to define the sister chromatid architecture during the cell cycle.

The most striking features that was revealed by the SISTERS project was the fact that sister chromatid cohesion in bacteria is a two step process, controlled successively by DNA topological links and protein mediated anchoring to the cytoskeleton.

Characterization of the molecular details controlling sister chromatid cohesion. Identification of some yet uncharacterized partners. Analysis of the influence of the sister chromatid cohesion step on DNA metabolisms reactions such as gene expression control and DNA lesion repair.

Lesterlin C, Gigant E, Boccard F, Espeli O, (2012) Sister chromatid interactions in bacteria revealed by a site specific recombination assay EMBO J, August 15. 31:3468-79

Espeli O, Borne R, Dupaigne P, Thiel A, Gigant E, Mercier R, Boccard F (2012) A MatP-divisome interaction coordinates chromosome segregation with cell division in E. coli. EMBO J May 11. 31:3198-11

Sister-chromatid cohesion is an essential step of the cell cycle of eukaryotic cells. This step has recently been described in bacteria, however its role and determinants remain poorly characterized. In Eukaryotic cells, establishment of the cohesion is dependent upon loading of cohesin proteic complexes on the sister-chromatid following replication. In bacteria, cohesines are not present and a combination of proteic factors and DNA untangling are believed to promote cohesion. Currently, the extent of the cohesion step is measured in term of co-localization of two sister tags. The method is very imprecise because it requires artifact prone fluorescent chromosome tagging and measures of the cell cycle parameters. We have developed, in E. coli, an alternative genetic assay allowing to measure distance between sister-chromatids in a more direct way. This assay revealed that sister-chromatid remained in close proximity for short period following replication. We called this step molecular cohesion. The extent of molecular cohesion differs from that of sister foci co-localization. The extent of molecular cohesion is dependent upon the considered locus. Topoisomerases activity, seems to modulate the extent of the cohesion period. Using this assay we have been able to identify a new protein involved in the cohesion step (SciA). The proposal for the ANR JC grant call focuses on four aspects: i) the characterization of the putative involvement of topological links in the establishment of sister-chromatids cohesion; ii) the characterization of the DNA determinants for sister-chromatids cohesion with a genome wide approach; iii) the identification of the role of the newly identified CccA protein for sister chromatid cohesion; iv) the development of cellular biology methods dedicated to the observation of the sister-chromatids. High-throughput sequencing technology will be combined with sophisticated genetic and cell biology tools to describe sister-chromatid organization during E. coli cell cycle, the proteins involved in the process and finally to tackle the role of the cohesion step in the chromosome metabolism.