Study of post-translational modifications of poplymerase eta

Post translational modifications (PTMs) play and important role in regulating TLS. Archetypical of this regulation is polη that has been shown to be ubiquitylated, phosphorylated, SUMoylated and O-GlcNAcylated. We were among the firsts to identify ubiquitylation and phosphorylation as two important signals that govern the roles of the polymerase, modulating its recruitment to stalled replication forks, the activation of damage bypass and the protein stability during the cell cycle. Unfortunately, we still do not completely know how these modifications are established and reversed and the repercussions on polη activity. This project tries to address these questions by evaluating the PTMs affecting the polymerase by 2D PAGE (see picture) and assessing the dynamics of the replication fork in mutants of the residues that are modified. 

 

Fig1. 2D-PAGE gel

We assess the fate and stability of the replication fork by DNA fibre analysis and evaluate the survival of the cells in different experimental conditions. In addition, we try to identify what proteins are involved in the regulation of PTMs and how they affect the activity of polη and the TLS pathway as a whole.

 

Fig2. Fork progression analysis by DNA fibres.