Replication, transcription, and translation stress all lead to stalling of their respective polymerases (DNA polymerase, RNA polymerase, and the ribosome), and the cell must respond to these events in order to preserve macromolecular integrity. In response to replication stress such as DNA damage, the cell activates a checkpoint and promotes repair or bypass at the lesion. Transcriptional stress leading to stalling of RNA polymerase can also be caused by DNA damage, and recognizing stalled RNA polymerase can lead to transcription-coupled repair or, in response to prolonged stalling, degradation of the polymerase. Translational stress generated by problems either with the mRNA template or the ribosome itself also leads to stalling of the ribosome, and the cell responds by degrading both the message and the nascent polypeptide. In this review, we will discuss the stresses that lead to stalling of each of the polymerases and how the cell recognizes and responds to the stalled enzymes.

The 33 genes in the Saccharomyces cerevisiae mitotic CLB2 transcription cluster have been known to be downregulated by the DNA damage checkpoint for many years. Here, we show that this is mediated by the checkpoint kinase Rad53 and the dedicated transcriptional activator of the cluster, Ndd1. Ndd1 is phosphorylated in response to DNA damage, which blocks recruitment to promoters and leads to the transcriptional downregulation of the CLB2 cluster. Finally, we show that downregulation of Ndd1 is an essential function of Rad53, as a hypomorphic ndd1 allele rescues RAD53 deletion. Over a decade ago, a group of mitotic genes called the CLB2 cluster was shown to be transcriptionally downregulated in response to DNA damage (1). The CLB2 cluster consists of 33 coregulated mitotic genes, including those for its namesake, Clb2 (a B-type cyclin), Cdc5 (Polo kinase), Cdc20 (the activator of the anaphase promoting complex), Hst3 (a sirtuin histone deacetylase), and many others (3). Because so many important mitotic regulators are part of this cluster, it is a hub for the regulation of mitosis during the cell cycle and in response to DNA damage.In an unperturbed cell cycle, CLB2 cluster transcription is tightly regulated, with transcription off during G 1 phase and high in early mitosis (3-5). Throughout the cell cycle, Mcm1 and Fkh2 are present at the promoters of these mitotic genes and coordinate both repression and activation by recruiting additional transcriptional regulators (4, 5). During G 1 , transcription of this cluster is off. As cells enter S phase, Clb5-cyclin-dependent kinase (CDK) phosphorylates Fkh2 (6). Later, Clb2-CDK and Cdc5 phosphorylate Ndd1, which leads to Ndd1's recruitment to its target promoters through an interaction with Fkh2 (7,8,27). Ndd1 then functions as a transcriptional activator to drive high levels of CLB2 cluster transcription (4,8). Ndd1 is itself cell cycle regulated and is transcribed in early S phase (9, 10). As both CLB2 and CDC5 are CLB2 cluster members themselves, these phosphorylations generate a positive-feedback loop that drives switch-like transcription of the cluster. In addition, the precise timing of transcription of this cluster is modulated by protein kinase C (PKC) (11) and, for a subset of members, Yox1 (12, 13).In response to DNA damage agents, transcription of the CLB2 cluster is downregulated by the DNA damage checkpoint (1, 2). This checkpoint is a signal transduction cascade that is activated in response to genotoxic stress, and the checkpoint is required to prevent replication fork collapse and arrest the cell cycle (14, 15). At the top of the kinase cascade that makes up the checkpoint, the phosphatidylinositide 3-kinase-like kinase Mec1 (the homolog of human ATR) is activated. Mec1 then phosphorylates and activates downstream effector kinases Chk1 and Rad53 (homologs of human Chk1 and Chk2, respectively) (16). Chk1 has a well-described role in promoting cell cycle arrest by phosphorylating and thereby stabilizing Pds1 (the Saccharomyces cerevisiae securin) (17-19...

Hst3 is the histone deacetylase that removes histone H3K56 acetylation. H3K56 acetylation is a cell-cycle-and damage-regulated chromatin marker, and proper regulation of H3K56 acetylation is important for replication, genomic stability, chromatin assembly, and the response to and recovery from DNA damage. Understanding the regulation of enzymes that regulate H3K56 acetylation is of great interest, because the loss of H3K56 acetylation leads to genomic instability. HST3 is controlled at both the transcriptional and posttranscriptional level. Here, we show that Hst3 is targeted for turnover by the ubiquitin ligase SCF Cdc4 after phosphorylation of a multisite degron. In addition, we find that Hst3 turnover increases in response to replication stress in a Rad53-dependent way. Turnover of Hst3 is promoted by Mck1 activity in both conditions. The Hst3 degron contains two canonical Cdc4 phospho-degrons, and the phosphorylation of each of these is required for efficient turnover both in an unperturbed cell cycle and in response to replication stress.H istone H3K56 acetylation is a cell-cycle-and damageregulated histone modification (1). In Saccharomyces cerevisiae, K56 acetylation is important for replication, genomic stability, chromatin assembly, and the response and recovery from DNA damage (1-6). Because of the critical nature of this chromatin mark, the enzymes that control it are themselves very important.In the budding yeast S. cerevisiae, Hst3 is a sirtuin histone deacetylase that, along with its close homolog Hst4, removes K56 acetylation (5, 7, 8). Overexpression of Hst3 or deletion of Hst3, along with related deacetylase Hst4, sensitizes cells to replication stress, suggesting that cells must control Hst3 levels precisely (5, 7). Hst3 is regulated both transcriptionally and by protein turnover. During an unperturbed cell cycle, HST3 transcript levels cycle, and Hst3 protein turns over very quickly (5,9). In response to DNA damage, transcription of HST3 is turned down, and Hst3 protein turnover is even faster (5,8,(10)(11)(12).Many cell-cycle-regulated proteins are targeted for proteasomal degradation after ubiquitination by a member of the SCF E3 ubiquitin ligase family. SCF ligases are multisubunit ubiquitin ligases composed of Skp1, a cullin (Cdc53), a RING-finger protein (Rbx1), and an F-box protein (13). The F-box protein is the substrate recognition module that confers substrate specificity to SCF ligases, with different F-box proteins recognizing different sets of substrates. The essential F-box protein Cdc4 regulates many cell-cycle-regulated proteins after their phosphorylation, including Sic1, Eco1, Cln3, and many others (14-19).Here, we have characterized the mechanism of Hst3 protein turnover. We find that Hst3 is targeted for degradation by SCF Cdc4 through a multisite phospho-degron. We find that Hst3 turnover is increased in response to replication stress in a Rad53-dependent way. Finally, we show that stabilizing Hst3 leads to misregulation of K56 acetylation in response to DNA damage and that...

In Saccharomyces cerevisiae, Ndd1 is the dedicated transcriptional activator of the mitotic gene cluster, which includes thirty-three genes that encode key mitotic regulators, making Ndd1 a hub for the control of mitosis. Previous work has shown that multiple kinases, including cyclin-dependent kinase (Cdk1), phosphorylate Ndd1 to regulate its activity during the cell cycle. Previously, we showed that Ndd1 was inhibited by phosphorylation in response to DNA damage. Here, we show that Ndd1 is also subject to regulation by protein turnover during the mitotic cell cycle: Ndd1 is unstable during an unperturbed cell cycle, but is strongly stabilized in response to DNA damage. We find that Ndd1 turnover in metaphase requires Cdk1 activity and the ubiquitin ligase SCFGrr1. In response to DNA damage, Ndd1 stabilization requires the checkpoint kinases Mec1/Tel1 and Swe1, the S. cerevisiae homolog of the Wee1 kinase. In both humans and yeast, the checkpoint promotes Wee1-dependent inhibitory phosphorylation of Cdk1 following exposure to DNA damage. While this is critical for checkpoint-induced arrest in most organisms, this is not true in budding yeast, where the function of damage-induced inhibitory phosphorylation is less well understood. We propose that the DNA damage checkpoint stabilizes Ndd1 by inhibiting Cdk1, which we show is required for targeting Ndd1 for destruction.

In this issue of Molecular Cell, Ohouo et al. (2010) show that Mec1 (hATR) promotes the association of Slx4 and Rtt107 with Dpb11 (hTopBP1) in response to MMS-induced DNA alkylation, suggesting that Slx4 and Rtt107 might coordinate repair factors specifically at damaged replication forks.