The DNA Replication Checkpoint Directly Regulates MBF-Dependent G₁/S Transcription

Abstract

The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G 1 /S transcriptional program by directly regulating MBF, the G 1 /S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G 1 /S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G 1 /S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes.In response to inhibition of DNA replication, the replication checkpoint arrests the cell cycle, stabilizes replication forks, and regulates transcription (3, 5). The transcriptional branch of the checkpoint response upregulates genes thought to be important for cells to survive prolonged replication arrest and to synthesize extra deoxynucleotides. A commonly used trigger of the replication checkpoint is treatment with the ribonucleotide reductase inhibitor hydroxyurea (HU), which prevents deoxynucleotide synthesis, thus preventing DNA replication. HU treatment activates a conserved checkpoint signaling pathway (3). In the fission yeast Schizosaccharomyces pombe, the central checkpoint kinase Rad3 (the homolog of the budding yeast Mec1 and metazoan ATM and ATR) activates the Sphase checkpoint effector kinase Cds1 (the homolog of the budding yeast Rad53 and Dun1, also called Chk2 in mammals), which regulates the downstream targets of the checkpoint (3). In the budding yeast Saccharomyces cerevisiae, Rad53 and Dun1 are thought to control transcription mainly through the Rfx1/Crt1 transcriptional repressor, which regulates a replication and DNA damage checkpoint-specific transcriptional program (21). However, the Rfx1 regulatory circuit is not evolutionarily conserved and it is unclear how the replication checkpoint regulates transcription in other eukaryotes.During a normal fission yeast cell cycle, G 1 /S transcription is regulated by the MBF transcription factor, also known as DSC1, and its negative regulator Nrm1 (1, 10). MBF-including the proteins Cdc10, Res1, Res2, Rep1, and Rep2-and Nrm1 are conserved in budding yeast, which also contains the paralogou...

TOR Complex 2 Controls Gene Silencing, Telomere Length Maintenance, and Survival under DNA-Damaging Conditions

“…4B). This finding could reflect either a defect in cell cycle progression in ⌬tor1 mutants or a more direct defect in activating the transcriptional response to HU (5)(6)(7)46).…”

A functional genome-wide genetic screening identifies new pathways controlling the G1/S transcriptional wave

“…We generated different strains that either were expressing chimeras of a fluorescent protein (YFP or GFP) fused to MBF-regulated genes (Cdc22-YFP, Yox1-GFP or Tos4-GFP) or were expressing YFP directly under the control of MBF-regulated promoters (pcdc18, pcdt2, pmik1 or ptos4). 3,11,13,14 By microscopic observation, we could detect fluorescence in each of the 7 strains that we tested, but with different intensity. Since we wanted to establish a method that would allow quantifying this fluorescence, we decided to determine their intrinsic fluorescence on the cytometer.…”

A homeodomain transcription factor regulates the DNA replication checkpoint in yeast

“…This result is interesting MBF-dependent gene expression. 3,[20][21][22] Interestingly, one aspect of this regulation involves phosphorylation of the Nrm1 repressor by Cds1 which results in derepression of MBF-dependent gene expression. 13,23 Here, we have further investigated the potential role(s) and regulation of the Yox1 homeodomain transcription factor in S. pombe.…”

“…Previous work demonstrated that MBF-regulated gene expression is upregulated in response to activation of the Rad3-dependent DNA replication checkpoint. 3,[20][21][22] Hence, we next examined whether Yox1 plays any role(s) in Rad3 checkpoint-regulated expression of MBF-dependent genes. Cells lacking Rad3 function display increased sensitivity to hydroxyurea (HU) and ultraviolet (UV) light (Fig.…”

“…Phosphomimetic mutations of proposed Cds1 phosphorylation sites in the MBF component Cdc10 are sufficient to induce MBFdependent transcription. 22 Additionally, Cds1 has been proposed to phosphorylate the MBF co-repressor Nrm1 in order to promote its dissociation from promoters, and therefore derepress target gene expression. 23 The regulation of both Nrm1 and Cdc10 in this manner appears to represent a redundant mechanism, whereby the phosphorylation of either protein by Cds1 is sufficient for the checkpoint-mediated elevation of MBF-dependent gene expression.…”