Abstract
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. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers 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.
Highlights
To ensure the genome stability, DNA replication is under strict surveillance by the S-phase checkpoint in all eukaryotes [1,2,3,4,5]
The appropriate amount and balance of four deoxyribonucleotide triphosphates (dNTPs) are crucial for all cells correctly copying and passing on their genetic material generation by generation
RNR1 gene is activated during G1/S transition by the MBF transcription factor, while the excessive expression of RNR2-4 is repressed by Crt1 (Constitutive ribonucleotide reductase (RNR) Transcription 1, called Rfx1) through recruiting the Ssn6-Tup1 co-repressor complex to the promoter
Summary
To ensure the genome stability, DNA replication is under strict surveillance by the S-phase checkpoint ( known as the intra-S or replication checkpoint) in all eukaryotes [1,2,3,4,5]. Among all the various downstream effects, the essential role of Mec1-Rad has been demonstrated to be in regulation of the RNR activity in Saccharomyces cerevisiae [3, 11, 12]. The expression of RNR1-4 genes is controlled at both transcriptional and post-transcriptional levels. RNR1 gene is activated during G1/S transition by the MBF transcription factor, while the excessive expression of RNR2-4 is repressed by Crt (Constitutive RNR Transcription 1, called Rfx1) through recruiting the Ssn6-Tup co-repressor complex to the promoter. The RNR enzyme activity is post-translationally inhibited by several small intrinsically disordered proteins such as Sml, Dif, and Hug in S. cerevisiae and Spd in Schizosaccharomyces pombe [15]. Like Dif, contains a HUG domain, which can inhibit RNR through binding Rnr2 [24, 25]
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