Mck1 defines a key S-phase checkpoint effector in response to various degrees of replication threats

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. As a kinase at the downstream of Rad53, Mck1 does not participate in the post-translational regulation of RNR as Dun1 does, but Mck1 can release the Crt1 repressor from the promoters of RNR2/3/4 by phosphorylation. Meanwhile, Hug1, an Rnr2 inhibitor, is induced to fine-tune the dNTP levels. When cells suffer a more severe threat, Mck1 can inhibit the transcription of HUG1. Importantly, only a combined deletion of HUG1 and CRT1, can confer a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis. Author Summary The appropriate amount and balance of four dNTPs are crucial for all cells correctly copying and passing on their genetic material generation by generation. Eukaryotes have developed an alert and response system to deal with the disturbance. Here, we uncovered a second-level effector branch. It is activated by the upstream surveillance kinase cascade, which can induce the expression of dNTP-producing enzymes. It can also reduce the inhibitor of these enzymes to further boost their activity according to the degrees of threats. These findings suggest a multi-level response system to guarantee dNTP supply, which is essential to maintain genetic stability under various environmental challenges.

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