A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress

Abstract

Upon replication stress, budding yeast checkpoint kinase Mec1 ATR 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.

A DNA damage-induced phosphorylation circuit enhances Mec1ATR-Ddc2ATRIPrecruitment to Replication Protein A

“…Since Rfa1-S178 phosphorylation influences Mec1-Ddc2 association, we next asked if phosphorylation of Mec1-Ddc2 can also influences recruitment. Proteomic and genetic studies have demonstrated a number of phosphorylation sites in Mec1-Ddc2 that are important for function (Hustedt et al, 2015;Memisoglu et al, 2019). We found that purified Mec1-Ddc2 used in our previous studies is extensively phosphorylated and the majority of Ddc2's phosphorylation can be removed by phosphatase treatment (Supplementary Figure 3A).…”

“…Both Mec1 and Ddc2 are extensively phosphorylated in response to DNA damage, replication stress, and during the cell cycle, in a Mec1-dependent and -independent manner, and are suggested to regulate the checkpoint (Bastos de Oliveira et al, 2015;Hustedt et al, 2015;Memisoglu et al, 2019;Paciotti et al, 2000). Two characterised Ddc2 phosphosites, S173 and S182, have been shown to permanently arrest the cell cycle in response to a DSB when mutated to an alanine (Memisoglu et al, 2019).…”

“…The flexible linker in Ddc2 would also allow a range of motion to enable Mec1 to phosphorylate many substrates, or other Mec1-Ddc2 complexes in our multivalent assembly. This arrangement will also likely promote Mec1 autophosphorylation, which occurs in trans , and is shown to be important for Mec1 function and repair efficiency (Hurst et al, 2021; Memisoglu et al, 2019; Sanford et al, 2021).…”

“…Given that a number of Mec1 activators are associated with chromatin, controlled recruitment of Mec1-Ddc2 by post-translational modifications (PTMs) could also be a means to regulate its cellular activity (Dubois et al, 2017; Maréchal et al, 2014; Memisoglu et al, 2019). Autophosphorylation of Mec1 is also important for its function (Hurst et al, 2021; Sanford et al, 2021) and occurs in trans (Memisoglu et al, 2019) implying that Mec1-Ddc2 must be brought together for this to occur. The localised Mec1 response at the DNA lesion site, characterised by H2A S129 phosphorylation, is rapid and hypersensitive to low levels of ssDNA (Bantele et al, 2019).…”

Coupling DNA Replication and Spindle Function in Saccharomyces cerevisiae

“…For example, the differences between wild-type spindles and spindles that elongate precociously in the absence of the S-phase checkpoint have not been studied in detail. Additional MT- and kinetochore-associated factors, apart from the few already known, seem to be phosphorylated by the S-phase checkpoint [ 67 ], with an unknown outcome. An open issue is also the force balance that prevents metaphase spindles from elongating in absence of CEN duplication under replication stress.…”