Periodic Reporting for period 4 - ChromatidCohesion (Establishment of Sister Chromatid Cohesion)
Okres sprawozdawczy: 2020-04-01 do 2022-03-31
The blueprint of our life is the DNA that stores all the information needed to build cells and organisms. Because of the sheer amount of information, DNA molecules are exceedingly long. At the time when human cells divide, they contain 4 metres of DNA, packed into micrometre-sized chromosomes. We are addressing how so much DNA can be arranged in such a small space. DNA does not come alone, it is bound by proteins. One of the prominent proteins on mitotic DNA is the chromosomal 'cohesin' complex. These are ring-shaped proteins that bind to DNA by topological embrace. We think that cohesin can bind to more than one DNA, such that it can establish interactions between distant parts of the genome. It also establishes interactions between the two copies of the genome that are replicated during S-phase and holds them together as what we call 'sister chromatids' until the time for the segregation towards daughter cells during cell division.
Because of their fundamental contribution to DNA organisation within the cell nucleus and within chromosomes, cohesin impinges on almost all processes that happen on DNA. These include gene transcription, DNA repair, chromosome compaction and chromosome segregation. Also, because of this, mutations in cohesin or its regulators are responsible for a large range of human illnesses. These range from severe developmental disorders to tumourigensis.
Our aim is to provide molecular insight into the function of the cohesin complex, so that we understand how cells work with their genomes, both in health and disease.
Because of their fundamental contribution to DNA organisation within the cell nucleus and within chromosomes, cohesin impinges on almost all processes that happen on DNA. These include gene transcription, DNA repair, chromosome compaction and chromosome segregation. Also, because of this, mutations in cohesin or its regulators are responsible for a large range of human illnesses. These range from severe developmental disorders to tumourigensis.
Our aim is to provide molecular insight into the function of the cohesin complex, so that we understand how cells work with their genomes, both in health and disease.
- We have biochemically resontituted the behaviour of the cohesin ring using purified components in vitro. This has led us to come up with a molecular model how DNA enters and exits from the cohesin ring.
- We have repeated the biochemical reconstitution using proteins from two different yeast species. This has shown the evolutionary conservation of the process, but also revealed important differences.
- We have characterised the role of a critical component of the DNA replication fork, Chl1, and its contribution to sister chromatid cohesion establishment by directly physically contacting the cohesin rings during DNA replication.
- To visualise the conformational dynamics of the cohesin ring, we have constructed variant cohesin complexes that have fluorophores attached at defined locations. We have begun to watch their conformational dynamics by both bulk and single molecule observations.
- We have uncovered how a chromatin remodeler plays a two-fold role in loading cohesin onto DNA. It acts as both, the chromatin receptor for the cohesin loader complex, as well as an active machine that generates a nucleosome-free region that is required as the substrate for cohesin loading.
- We have repeated the biochemical reconstitution using proteins from two different yeast species. This has shown the evolutionary conservation of the process, but also revealed important differences.
- We have characterised the role of a critical component of the DNA replication fork, Chl1, and its contribution to sister chromatid cohesion establishment by directly physically contacting the cohesin rings during DNA replication.
- To visualise the conformational dynamics of the cohesin ring, we have constructed variant cohesin complexes that have fluorophores attached at defined locations. We have begun to watch their conformational dynamics by both bulk and single molecule observations.
- We have uncovered how a chromatin remodeler plays a two-fold role in loading cohesin onto DNA. It acts as both, the chromatin receptor for the cohesin loader complex, as well as an active machine that generates a nucleosome-free region that is required as the substrate for cohesin loading.
In several of our workpackages, we have gone beyond the current state of the art. We were the first ones to biochemically reconstitute the topological DNA loading reaction of the cohesin ring. We have now achieved this using proteins from a second, evolutionarily distant, yeast species. We have meanwhile also reconstituted the unloading reaction and we are currently the first ones to perform single molecule conformational observations with this complex.