Abstract
BackgroundMechanisms 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.ResultsHere, 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.ConclusionsBy 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.
Highlights
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
Studies conducted in S. cerevisiae showed redundancy between Exo1 and Rad27 in processing Okazaki fragments during DNA replication [20] and the recruitment of yeast Exo1 to stalled replication forks was shown to contribute to fork stability by counteracting fork reversal [21]
Taking advantage of the superior performance of Phos-Tag [30] as compared to regular SDS-PAGE [31] in resolving phosphorylated forms of Exo1, we first visualized the effect of DNA damage by an alkylating agent or of stalled DNA replication on Exo1 mobility (Fig. 1a and Additional file 1: Fig. S1)
Summary
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. A role in mutation avoidance and mismatch correction was described for S. pombe Exo1 [13] and later confirmed in S. cerevisiae, demonstrating physical and genetic interaction between yeast Exo and the DNA mismatch repair (MMR) proteins Msh2 [14] and Mlh1 [15]. Both S. cerevisiae and human EXO1 were shown to participate in the process of nucleotide excision repair (NER) after UV irradiation [16, 17]. Studies conducted in S. cerevisiae showed redundancy between Exo and Rad in processing Okazaki fragments during DNA replication [20] and the recruitment of yeast Exo to stalled replication forks was shown to contribute to fork stability by counteracting fork reversal [21]
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