Periodic Reporting for period 1 - RESTART (The Role of cohEsin dynamicS at sTAlled ReplicaTion forks)
Okres sprawozdawczy: 2023-11-01 do 2025-10-31
Cohesin, an evolutionary conserved ring-shaped protein complex that topologically entraps DNA, has crucial roles in many structural and functional aspects of chromosomes including sister chromatid cohesion, genome organization, gene transcription and DNA repair. The importance of cohesin in the maintenance of genomic stability has been known for some time. Cohesin facilitates double strand breaks repair by homologous recombination and promotes the restart of stalled replication forks by template switching, an error-free DNA tolerance pathway. However, the precise role of cohesin in DNA damage and in the recovery after replication stress is still unknown. Whether cohesin acts exclusively providing close proximity of sister chromatids or its functions exceed simple embracing of DNA has yet to be discerned. Cohesin has been shown to be ubiquitylated upon replication fork arrest. This post-translational modification has been proposed to favour cohesin mobilization and subsequent association with nascent DNA behind the fork. However, the extent to what cohesin dynamics are important in the recovery after replication stress is still to be understood. By a range of multidisciplinary approaches, from biochemistry and enzymology, yeast genetics and molecular biology techniques to high throughput proteomics and bioinformatics analysis of mass spectrometry data, we will gain valuable insight about the interplay between sister chromatid cohesion and DNA damage tolerance and their contribution to successful replication. This basic knowledge about the mechanisms involved in DNA damage responses has demonstrated to be essential in the design of successful strategies against cancer. Remarkably, cohesin is one of the protein complexes most frequently mutated in cancer. This project aims to provide valuable insight about how cohesin mutations cause tumorigenesis.
RESTART action has been preliminary ended because Sofía Muñoz Félix, the MSCA Fellow, got a principal investigator position. This fact evidences the positive impact that the Action has had in the scientific career of the Fellow. However, even though important advance towards the objectives defined in the proposal has been made, the work packages proposed have not been completed. Nevertheless, the progress made towards the objectives will be used in future investigations in the research group of the Fellow.
Cohesin was initially described and is broadly renowned for its essential role in holding sister chromatids together following DNA replication, which is necessary for faithful chromosome segregation during cell divisions. However, in spite of the vast knowledge about the biology of the cohesin complex acquired during the last decades and much progress has been made in understanding the role of cohesin in the DNA damage response, the precise role of cohesin in DNA damage and restart of stalled replication forks is still unknown. Cohesin implication in fork restart after damage has been studied using cohesin thermosensitive mutants, defective for all cohesin functions. On the other hand, cohesin ubiquitylation importance for DNA damage tolerance has been inferred from the fact that mutants in the ubiquitin ligase Rps5 or its adaptors are defective in stalled fork progression in an epistatic way with cohesin thermosensitive alleles. RESTART proposes to identify and mutate cohesin ubiquitylation sites, which will allow me to study the importance of this post-translational modification in the recovery after replication fork arrest in a direct manner and most importantly, getting separation of function mutants, proficient for sister chromatid cohesion but impaired in the DNA damage response, will permit for the first time to discern whether the specific role of cohesin in DNA damage tolerance is independent of its role in cohesion. In addition, this problem has never been addressed from a biochemical point of view. The biochemical reconstitution of reactions that happen inside the cell has proven to be a very valuable tool to understand the molecular mechanisms underlying biological processes. The reconstitution of the cohesin ubiquitylation reaction not only will provide evidence of the requirements for cohesin ubiquitylation and allow me to study its regulation but also will allow me to analyze the biochemical behavior of ubiquitylated cohesin.