1. A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress
- Author
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Naama Barkai, Romain Forey, Kiran Challa, Jan Seebacher, Susan M. Gasser, Felix Jonas, Kenji Shimada, Jérôme Poli, Christoph D. Schmid, Ragna Sack, and Verena Hurst
- Subjects
DNA Replication ,Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,replication stress ,DNA Replication, Recombination & Repair ,Saccharomyces cerevisiae ,Protein Serine-Threonine Kinases ,General Biochemistry, Genetics and Molecular Biology ,Article ,S Phase ,chemistry.chemical_compound ,Galactokinase ,Post-translational Modifications & Proteolysis ,Transcription (biology) ,Stress, Physiological ,RNA polymerase ,Gene Expression Regulation, Fungal ,Gene expression ,Hydroxyurea ,Mec1 ,Nuclear pore ,Phosphorylation ,Molecular Biology ,Gene ,Polymerase ,General Immunology and Microbiology ,biology ,General Neuroscience ,Intracellular Signaling Peptides and Proteins ,RNA ,RNA Polymerase III ,Articles ,Phosphoproteins ,replication interference ,Chromatin ,Cell biology ,chemistry ,nuclear pore ,Chromatin, Transcription & Genomics ,biology.protein ,replication checkpoint ,RNA Polymerase II ,transcription ,Protein Processing, Post-Translational - Abstract
Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea‐induced phosphorylation site on Mec1, Mec1‐S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non‐phosphorylatable mec1‐S1991A mutant reduces replication fork progression genome‐wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin‐bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1‐S1991A mutants arises from replication–transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1‐S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1‐dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit., Hydroxyurea directly triggers a checkpoint kinase Mec1/ATR function for limiting replication‐transcription conflicts in budding yeast.
- Published
- 2021