Cdk1-interacting protein Cip1 is regulated by the S phase checkpoint in response to genotoxic stress.

In eukaryotic cells, a surveillance mechanism, the S phase checkpoint, detects and responds to insults that challenge chromosomal replication, arresting cell cycle progression and triggering appropriate events to prevent genomic instability. In the budding yeast Saccharomyces cerevisiae, Mec1/ATM/ATR, and its downstream kinase, Rad53/Chk2, mediate the response to genotoxic stress. In this study, we place Cip1, a recently identified Cdk1 inhibitor (CKI), under the regulation of Mec1 and Rad53 in response to genotoxic stress. Cip1 accumulates dramatically in a Mec1- and Rad53-dependent manner upon replication stress. This increase requires the activity of MBF, but not the transcriptional activator kinase Dun1. At the protein level, stabilization of replication stress-induced Cip1 requires continued de novo protein synthesis. In addition, Cip1 is phosphorylated at an S/TQ motif in a Mec1-dependent manner. Deletion of Cip1 affects proliferation in hydroxyurea-containing plates. Significantly, the sensitivity is increased when the dosage of the G1 cyclin CLN2 is increased, compatible to a role of Cip1 as a G1-cyclin-dependent kinase inhibitor. In all, our results place Cip1 under the S phase checkpoint response to genotoxic stress. Furthermore, Cip1 plays a significant role to preserve viability in response to insults that threaten chromosome replication.

Three Different Pathways Prevent Chromosome Segregation in the Presence of DNA Damage or Replication Stress in Budding Yeast.

A surveillance mechanism, the S phase checkpoint, blocks progression into mitosis in response to DNA damage and replication stress. Segregation of damaged or incompletely replicated chromosomes results in genomic instability. In humans, the S phase checkpoint has been shown to constitute an anti-cancer barrier. Inhibition of mitotic cyclin dependent kinase (M-CDK) activity by Wee1 kinases is critical to block mitosis in some organisms. However, such mechanism is dispensable in the response to genotoxic stress in the model eukaryotic organism Saccharomyces cerevisiae. We show here that the Wee1 ortholog Swe1 does indeed inhibit M-CDK activity and chromosome segregation in response to genotoxic insults. Swe1 dispensability in budding yeast is the result of a redundant control of M-CDK activity by the checkpoint kinase Rad53. In addition, our results indicate that Swe1 is an effector of the checkpoint central kinase Mec1. When checkpoint control on M-CDK and on Pds1/securin stabilization are abrogated, cells undergo aberrant chromosome segregation.

A role for the spindle assembly checkpoint in the DNA damage response.

Spontaneous DNA damage poses a continuous threat to genomic integrity. If unchecked, genotoxic insults result in genomic instability, a hallmark of cancer cells. In eukaryotic cells a DNA Damage Response (DDR) detects and responds to genotoxic stress, acting as an anti-cancer barrier in humans. Among other actions, the DDR blocks the segregation of incompletely replicated or damaged chromosomes, thus preventing aneuploidy. In a work aimed at better understanding such S-M control, we recently showed that cells block anaphase through different control pathways. The S phase checkpoint kinase Mec1/ATR inhibits mitotic Cyclin Dependent Kinase activity through effector kinases Swe1/Wee1 and Rad53/Chk2. Cells also stabilize the levels of Pds1/securin to block sister chromatid segregation in response to DNA damage. We show here that Pds1/securin abundance is still secured when the S phase checkpoint response is fully abrogated in mec1/ATR tel1/ATM double null mutants. When such cells are exposed to genotoxic stress, Pds1/securin is stabilized in a spindle assembly checkpoint (SAC) dependent manner. Disruption of the SAC and the S phase checkpoint together, allows chromosome segregation in the presence of DNA damage or replication stress. Our results place the SAC as a part of the DDR, which appears to count on different, independent control layers to preserve genomic integrity when chromosome replication is challenged.

A Dbf4 mutant contributes to bypassing the Rad53-mediated block of origins of replication in response to genotoxic stress.

An intra-S phase checkpoint slows the rate of DNA replication in response to DNA damage and replication fork blocks in eukaryotic cells. In the budding yeast Saccharomyces cerevisiae, such down-regulation is achieved through the Rad53 kinase-dependent block of origins of replication. We have identified the Rad53 phosphorylation sites on Dbf4, the activator subunit of the essential S phase Dbf4-dependent kinase, and generated a non-phosphorylatable Dbf4 mutant (dbf4(7A)). We show here that dbf4(7A) is a bona fide intra-S phase checkpoint bypass allele that contributes to abrogating the Rad53 block of origin firing in response to genotoxic stress.

Cyclin regulation by the s phase checkpoint.

In eukaryotic cells a surveillance mechanism, the S phase checkpoint, detects and responds to DNA damage and replication stress, protecting DNA replication and arresting cell cycle progression. We show here that the S phase cyclins Clb5 and Clb6 are regulated in response to genotoxic stress in the budding yeast Saccharomyces cerevisiae. Clb5 and Clb6 are responsible for the activation of the specific Cdc28 cyclin-dependent kinase activity that drives the onset and progression of the S phase. Intriguingly, Clb5 and Clb6 are regulated by different mechanisms. Thus, the presence of Clb6, which is eliminated early in an unperturbed S phase, is stabilized when replication is compromised by replication stress or DNA damage. Such stabilization depends on the checkpoint kinases Mec1 and Rad53. The stabilization of Clb6 levels is a dynamic process that requires continued de novo protein synthesis, because the cyclin remains subject to degradation. It also requires the activity of the G(1) transcription factor Mlu1 cell cycle box-binding factor (MBF) in the S phase, whereas Dun1, the checkpoint kinase characteristically responsible for the transcriptional response to genotoxic stress, is dispensable in this case. On the other hand, two subpopulations of endogenous Clb5 can be distinguished according to turnover in an unperturbed S phase. In the presence of replication stress, the unstable Clb5 pool is stabilized, and such stabilization requires neither MBF transcriptional activity nor de novo protein synthesis.

High-Copy Yeast Library Construction and High-Copy Rescue Genetic Screen in Saccharomyces cerevisiae.

High-copy rescue genetic screening is a powerful strategy for the identification of suppression genetic interactions in the model eukaryotic organism Saccharomyces cerevisiae (budding yeast). The strain carrying the mutant allele of interest is transformed with a genomic library cloned in a high-copy plasmid. Each clone carries a genomic fragment insertion of around 10 kb, typically containing one to three complete genes under their own promoters. The high-copy vector favors the accumulation of high levels of the corresponding protein, aimed at suppressing the mutant phenotype. Typically, high-copy genetic screens select for viable clones under conditions restrictive or lethal for the query mutant strain. Here, we describe in detail the procedure to generate a high-copy genomic library and a protocol for rescue genetic screening and identification of the suppressor clones.

Trafficking of Stretch-Regulated TRPV2 and TRPV4 Channels Inferred Through Interactomics.

Transient receptor potential cation channels are emerging as important physiological and therapeutic targets. Within the vanilloid subfamily, transient receptor potential vanilloid 2 (TRPV2) and 4 (TRPV4) are osmo- and mechanosensors becoming critical determinants in cell structure and activity. However, knowledge is scarce regarding how TRPV2 and TRPV4 are trafficked to the plasma membrane or specific organelles to undergo quality controls through processes such as biosynthesis, anterograde/retrograde trafficking, and recycling. This review lists and reviews a subset of protein-protein interactions from the TRPV2 and TRPV4 interactomes, which is related to trafficking processes such as lipid metabolism, phosphoinositide signaling, vesicle-mediated transport, and synaptic-related exocytosis. Identifying the protein and lipid players involved in trafficking will improve the knowledge on how these stretch-related channels reach specific cellular compartments.

G1 cyclin driven DNA replication.

The mitotic cell cycle is driven by Cyclin-Dependent Kinases (CDK). CDK activation requires the binding of activatory subunits termed cyclins. Different waves of cyclins are expressed during the cell cycle, enabling CDKs to trigger phase specific events. For instance, S phase cyclins promote the initiation of DNA replication but not chromosome segregation. There are at least 2 explanations for how such regulation is achieved. According to one of the visions, cyclins confer intrinsic substrate specificity to the CDK catalytic subunit. Alternatively a quantitative model has been proposed, according to which ever-increasing CDK activity is required to trigger cell cycle events from G1 to M. If a quantitative control prevails, then an early cyclin should trigger later cycle events if accumulated at high enough levels at the right time and place. We show here that a G1 phase cyclin bears the potential to trigger DNA replication and promote S and G2 phase specific transcription.

Distinct phosphatases mediate the deactivation of the DNA damage checkpoint kinase Rad53.

The DNA damage checkpoint regulates DNA replication and arrests cell cycle progression in response to genotoxic stress. In Saccharomyces cerevisiae, the protein kinase Rad53 plays a central role in preventing genomic instability and maintaining viability in the presence of replication stress and DNA damage. Activation of Rad53 depends on phosphorylation by the upstream kinase Mec1, followed by autophosphorylation on multiple residues. Also critical for cell viability, the molecular mechanism of Rad53 deactivation remains incompletely understood. Rad53 dephosphorylation after repair of a persistent double strand break in G(2)/M has been shown to depend on the presence of the PP2C-type phosphatases Ptc2 and Ptc3. More recently, the PP2A-like protein phosphatase Pph3 has been shown to be required to dephosphorylate Rad53 after DNA methylation damage in S phase. However, we show here that Ptc2/3 are dispensable for Rad53 deactivation after replication stress or DNA methylation damage. Pph3 is also dispensable for the deactivation of Rad53 after replication stress. In addition, Rad53 kinase activity is still deactivated in pph3 null cells after DNA methylation damage, despite persistent Rad53 hyperphosphorylation. Finally, a strain in which the three phosphatases are deleted shows a severe defect in Rad53 kinase deactivation after DNA methylation damage but not after replication stress. In all, our results suggest that distinct phosphatases operate to return Rad53 to its basal state after different genotoxic stresses and that a yet unidentified phosphatase may be responsible for the deactivation of Rad53 after replication stress.