Replication stress-induced Exo1 phosphorylation is mediated by Rad53/Pph3 and Exo1 nuclear localization is controlled by 14-3-3 proteins

Mechanisms controlling DNA resection at sites of damage and affecting genome stability have been the subject of deep investigation, though their complexity is not yet fully understood. Specifically, the regulatory role of post-translational modifications in the localization, stability and function of DNA repair proteins is an important aspect of such complexity.

By shedding light on the mechanism of Exo1 control, these data underscore the importance of post-translational modifications and protein interactions in the regulation of DNA end resection.

Here, we took advantage of the superior resolution of phosphorylated proteins provided by Phos-Tag technology to study pathways controlling the reversible phosphorylation of yeast Exo1, an exonuclease involved in a number of DNA repair pathways. We report that Rad53, a checkpoint kinase downstream of Mec1, is responsible for Exo1 phosphorylation in response to DNA replication stress and we demonstrate a role for the type-2A protein phosphatase Pph3 in the dephosphorylation of both Rad53 and Exo1 during checkpoint recovery. Fluorescence microscopy studies showed that Rad53-dependent phosphorylation is not required for the recruitment or the release of Exo1 from the nucleus, whereas 14-3-3 proteins are necessary for Exo1 nuclear translocation. Conclusions: By shedding light on the mechanism of Exo1 control, these data underscore the importance of post-translational modifications and protein interactions in the regulation of DNA end resection.