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.

中文翻译：

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Mec1 上的调节磷酸化位点控制复制应激期间 RNA 聚合酶的染色质占用

在复制压力下，出芽酵母检查点激酶 Mec1 ^ATR触发转录下调，从而降低染色质上 RNA 聚合酶 (RNAP) 的水平，以促进复制叉进展。在这里，我们确定了 Mec1 上的羟基脲诱导的磷酸化位点 Mec1-S1991，这有助于在复制应激期间驱逐 RNAPII 和 RNAPIII。不可磷酸化的mec1-S1991A突变体的表达降低了全基因组复制叉的进展，并损害了羟基脲的存活率。通过与 Rpb3 亚基的 TAP 融合使染色质结合的 RNAPII 不稳定，可以抑制这种缺陷，这表明mec1-S1991A 中的致死率突变体源于复制-转录冲突。与未能抑制mec1-S1991A细胞中羟基脲基因表达的同时，高度转录的基因（如GAL1）仍然结合在核孔中。一致地，我们发现核孔蛋白和控制 RNAPII 和 RNAPIII 的因子在羟基脲上以 Mec1 依赖性方式磷酸化。此外，我们表明 Mec1 激酶还通过促进 Rpb1 亚基的降解，在未受干扰的 S 期期间减少染色质上的 RNAPII 占有率。