Fission yeast Cdc14-like phosphatase Flp1/Clp1 modulates the transcriptional response to oxidative stress.

Reactive oxygen species (ROS) are an important source of cellular damage. When ROS intracellular levels increase, oxidative stress takes place affecting DNA stability and metabolic functions. To prevent these effects, stress-activated protein kinases (SAPKs) delay cell cycle progression and induce a transcriptional response that activates antioxidant mechanisms ensuring cell adaptation and survival.

Working on Genomic Stability: From the S-Phase to Mitosis.

Fidelity in chromosome duplication and segregation is indispensable for maintaining genomic stability and the perpetuation of life. Challenges to genome integrity jeopardize cell survival and are at the root of different types of pathologies, such as cancer. The following three main sources of genomic instability exist: DNA damage, replicative stress, and chromosome segregation defects.

PCNA Deubiquitylases Control DNA Damage Bypass at Replication Forks.

DNA damage tolerance plays a key role in protecting cell viability through translesion synthesis and template switching-mediated bypass of genotoxic polymerase-blocking base lesions. Both tolerance pathways critically rely on ubiquitylation of the proliferating-cell nuclear antigen (PCNA) on lysine 164 and have been proposed to operate uncoupled from replication. We report that Ubp10 and Ubp12 ubiquitin proteases differentially cooperate in PCNA deubiquitylation, owing to distinct activities on PCNA-linked ubiquitin chains.

Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1.

Cdc14 enzymes compose a family of highly conserved phosphatases that are present in a wide range of organisms, including yeast and humans, and that preferentially reverse the phosphorylation of Cyclin-Dependent Kinase (Cdk) substrates. The budding yeast Cdc14 orthologue has essential functions in the control of late mitosis and cytokinesis. In mammals, however, the two Cdc14 homologues, Cdc14A and Cdc14B, do not play a prominent role in controlling late mitotic events, suggesting that some Cdc14 functions are not conserved across species.

The Multiple Roles of Ubiquitylation in Regulating Challenged DNA Replication.

DNA replication is essential for the propagation of life and the development of complex organisms. However, replication is a risky process as it can lead to mutations and chromosomal alterations. Conditions challenging DNA synthesis by replicative polymerases or DNA helix unwinding, generally termed as replication stress, can halt replication fork progression.

Orderly progression through S-phase requires dynamic ubiquitylation and deubiquitylation of PCNA.

Proliferating-cell nuclear antigen (PCNA) is a DNA sliding clamp with an essential function in DNA replication and a key role in tolerance to DNA damage by ensuring the bypass of lesions. In eukaryotes, DNA damage tolerance is regulated by ubiquitylation of lysine 164 of PCNA through a well-known control mechanism; however, the regulation of PCNA deubiquitylation remains poorly understood. Our work is a systematic and functional study on PCNA deubiquitylating enzymes (DUBs) in Schizosaccharomyces pombe.

Analysis of the tolerance to DNA alkylating damage in MEC1 and RAD53 checkpoint mutants of Saccharomyces cerevisiae.

Checkpoint response, tolerance and repair are three major pathways that eukaryotic cells evolved independently to maintain genome stability and integrity. Here, we studied the sensitivity to DNA damage in checkpoint-deficient budding yeast cells and found that checkpoint kinases Mec1 and Rad53 may modulate the balance between error-free and error-prone branches of the tolerance pathway. We have consistently observed that mutation of the RAD53 counterbalances error-free and error-prone branches upon exposure of cells to DNA damage induced either by MMS alkylation or by UV-radiation.

Human Cdc14A regulates Wee1 stability by counteracting CDK-mediated phosphorylation.

The activity of Cdk1-cyclin B1 mitotic complexes is regulated by the balance between the counteracting activities of Wee1/Myt1 kinases and Cdc25 phosphatases. These kinases and phosphatases must be strictly regulated to ensure proper mitotic timing. One masterpiece of this regulatory network is Cdk1, which promotes Cdc25 activity and suppresses inhibitory Wee1/Myt1 kinases through direct phosphorylation.

Reversal of PCNA ubiquitylation by Ubp10 in Saccharomyces cerevisiae.

Regulation of PCNA ubiquitylation plays a key role in the tolerance to DNA damage in eukaryotes. Although the evolutionary conserved mechanism of PCNA ubiquitylation is well understood, the deubiquitylation of ubPCNA remains poorly characterized. Here, we show that the histone H2B(K123) ubiquitin protease Ubp10 also deubiquitylates ubPCNA in Saccharomyces cerevisiae.

Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription.

Kinases and phosphatases regulate messenger RNA synthesis through post-translational modification of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 1). In yeast, the phosphatase Cdc14 is required for mitotic exit(2,3) and for segregation of repetitive regions(4).

Human Cdc14A becomes a cell cycle gene in controlling Cdk1 activity at the G₂/M transition.

Cdc14 belongs to a dual-specificity phosphatase family highly conserved through evolution that preferentially reverses CDK (Cyclin dependent kinases) -dependent phosphorylation events. In the yeast Saccharomyces cerevisiae, Cdc14 is an essential regulator of late mitotic events and exit from mitosis by counteracting CDK activity at the end of mitosis. However, many studies have shown that Cdc14 is dispensable for exiting mitosis in all other model systems analyzed.

Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B.

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division.

Regulation of tolerance to DNA alkylating damage by Dot1 and Rad53 in Saccharomyces cerevisiae.

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous factors that produce genome injuries and interfere with DNA replication. DNA integrity checkpoints coordinate this response by slowing cell cycle progression to provide time for the cell to repair the damage, stabilizing replication forks and stimulating DNA repair to restore the original DNA sequence and structure. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage.

Control of PCNA deubiquitylation in yeast.

Eukaryotes ubiquitylate the replication factor PCNA (proliferating-cell nuclear antigen) so that it tolerates DNA damage. Although, in the last few years, the understanding of the evolutionarily conserved mechanism of ubiquitylation of PCNA, and its crucial role in DNA damage tolerance, has progressed impressively, little is known about the deubiquitylation of this sliding clamp in most organisms. In the present review, we will discuss potential molecular mechanisms regulating PCNA deubiquitylation in yeast.

The Flp1/Clp1 phosphatase cooperates with HECT-type Pub1/2 protein-ubiquitin ligases in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe Flp1p serine-threonine phosphatase is required for the degradation of the mitotic inducer Cdc25p at the end of mitosis. Cdc25p degradation prevents Cdc2p-tyrosine 15 dephosphorylation and, thus, contributes to the timely inactivation of mitotic CDK-associated kinase activity. Both RING- and HECT-type protein-ubiquitin ligases are involved in Cdc25p destabilization.

Cds1 controls the release of Cdc14-like phosphatase Flp1 from the nucleolus to drive full activation of the checkpoint response to replication stress in fission yeast.

The Cdc14p-like phosphatase Flp1p (also known as Clp1p) is regulated by cell cycle-dependent changes in its subcellular localization. Flp1p is restricted to the nucleolus and spindle pole body until prophase, when it is dispersed throughout the nucleus, mitotic spindle, and medial ring. Once released, Flp1p antagonizes Cdc2p/cyclin activity by reverting Cdc2p-phosphorylation sites on Cdc25p.

Human Cdc14A reverses CDK1 phosphorylation of Cdc25A on serines 115 and 320.

Human Cdc14A is an evolutionary conserved dual-specificity protein phosphatase that reverses the modifications effected by cyclin-dependent kinases and plays an important role in centrosome duplication and mitotic regulation. Few substrates of Cdc14A have been identified, some of them with homologues in yeast that, in turn, are substrates of the Saccharomyces cerevisiae Cdc14 homologue, a protein phosphatase essential for yeast cell viability owing its role in mitotic exit regulation. Identification of the physiological substrates of human Cdc14A is an immediate goal in order to elucidate which cellular processes it regulates.

Limiting amounts of budding yeast Rad53 S-phase checkpoint activity results in increased resistance to DNA alkylation damage.

The Saccharomyces cerevisiae protein kinase Rad53 plays a key role in maintaining genomic integrity after DNA damage and is an essential component of the 'intra-S-phase checkpoint'. In budding yeast, alkylating chemicals, such as methyl methanesulfonate (MMS), or depletion of nucleotides by hydroxyurea (HU) stall DNA replication forks and thus activate Rad53 during S-phase. This stabilizes stalled DNA replication forks and prevents the activation of later origins of DNA replication.

Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork.

Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM-Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly.

Functional homology among human and fission yeast Cdc14 phosphatases.

Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis.

A role for the Cdc14-family phosphatase Flp1p at the end of the cell cycle in controlling the rapid degradation of the mitotic inducer Cdc25p in fission yeast.

The Schizosaccaromyces pombe protein Flp1p belongs to a conserved family of serine-threonine-phosphatases. The founding member of this family, Saccharomyces cerevisiae Cdc14p, is required for inactivation of mitotic CDKs and reversal of CDK mediated phosphorylation at the end of mitosis, thereby bringing about the M-G1 transition. Initial studies of Flp1p suggest that it may play a different role to Cdc14p.

Genes involved in the initiation of DNA replication in yeast.

Replication and segregation of the information contained in genomic DNA are strictly regulated processes that eukaryotic cells alternate to divide successfully. Experimental work on yeast has suggested that this alternation is achieved through oscillations in the activity of a serine/threonine kinase complex, CDK, which ensures the timely activation of DNA synthesis. At the same time, this CDK-mediated activation sets up the basis of the mechanism that ensures ploidy maintenance in eukaryotes.