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.

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.

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.

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.

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.

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.

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)).

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.

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.