Kinetochore Components Required for Centromeric Chromatin Assembly Are Impacted by Msc1 in .

Eukaryotic chromosome segregation requires a protein complex known as the kinetochore that mediates attachment between mitotic spindle microtubules and centromere-specific nucleosomes composed of the widely conserved histone variant CENP-A. Mutations in kinetochore proteins of the fission yeast lead to chromosome missegregation such that daughter cells emerge from mitosis with unequal DNA content. We find that multiple copies of Msc1-a fission yeast homolog of the KDM5 family of proteins-suppresses the temperature-sensitive growth defect of several kinetochore mutants, including and , as well as , , and , components of the Constitutive Centromere Associated Network (CCAN).

Microtubule dynamics decoded by the epigenetic state of centromeric chromatin.

Cell division with accurate chromosome segregation is fundamental to cell survival of all organisms. The precise molecular mechanisms that ensure accurate chromosome segregation are still being discovered using a variety of experimental systems and approaches. Microtubule attachment to the kinetochore is a prerequisite for mitotic progression, failure of which activates the spindle assembly checkpoint (SAC).

Escape from Mitotic Arrest: An Unexpected Connection Between Microtubule Dynamics and Epigenetic Regulation of Centromeric Chromatin in Schizosaccharomyces pombe.

Accurate chromosome segregation is necessary to ensure genomic integrity. Segregation depends on the proper functioning of the centromere, kinetochore, and mitotic spindle microtubules and is monitored by the spindle assembly checkpoint (SAC). In the fission yeast Schizosaccharomyces pombe, defects in Dis1, a microtubule-associated protein that influences microtubule dynamics, lead to mitotic arrest as a result of an active SAC and consequent failure to grow at low temperature.

The oxidative stress responsive transcription factor Pap1 confers DNA damage resistance on checkpoint-deficient fission yeast cells.

Eukaryotic cells invoke mechanisms to promote survival when confronted with cellular stress or damage to the genome. The protein kinase Chk1 is an integral and conserved component of the DNA damage response pathway. Mutation or inhibition of Chk1 results in mitotic death when cells are exposed to DNA damage.

A molecular evolution approach to study the roles of tropomyosin in fission yeast.

Tropomyosin, a coiled-coil protein that binds along the length of the actin filament, is a universal regulator of the actin cytoskeleton. We have taken a bioinformatics/proteomic approach to studying structure-function relationships in this protein. The presence of a single, essential tropomyosin gene, cdc8, in fission yeast, Schizosaccharomyces pombe, enables a systems-based approach to define the residues that are important for cellular functions.

Activity of a C-terminal plant homeodomain (PHD) of Msc1 is essential for function.

Msc1, a member of the Jarid1 family of putative histone demethylases, is required for chromosome stability in fission yeast. Msc1 associates with the Swr1 complex that facilitates deposition of histone H2A.Z into chromatin.

Importance of a C-terminal conserved region of Chk1 for checkpoint function.

Background: The protein kinase Chk1 is an essential component of the DNA damage checkpoint pathway. Chk1 is phosphorylated and activated in the fission yeast Schizosaccharomyces pombe when cells are exposed to agents that damage DNA. Phosphorylation, kinase activation, and nuclear accumulation are events critical to the ability of Chk1 to induce a transient delay in cell cycle progression.

Msc1 acts through histone H2A.Z to promote chromosome stability in Schizosaccharomyces pombe.

As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components.

The plant homeodomain fingers of fission yeast Msc1 exhibit E3 ubiquitin ligase activity.

The DNA damage checkpoint pathway governs how cells regulate cell cycle progression in response to DNA damage. A screen for suppressors of a fission yeast chk1 mutant defective in the checkpoint pathway identified a novel Schizosaccharomyces pombe protein, Msc1. Msc1 contains 3 plant homeodomain (PHD) finger motifs, characteristically defined by a C4HC3 consensus similar to RING finger domains.

Interaction of 14-3-3 protein with Chk1 affects localization and checkpoint function.

The protein kinase Chk1 is required for proper arrest of the cell cycle in response to DNA damage. We have previously shown in Schizosaccharomyces pombe, that upon DNA damage, phosphorylation of Chk1 correlates with checkpoint activation and that phosphorylated Chk1 is capable of interacting with the 14-3-3 proteins, Rad24 and Rad25. The interaction between Rad24 and Chk1 is stimulated tenfold after exposure to DNA damaging agents and we postulate that it is an important event in the DNA damage checkpoint response pathway in fission yeast.

Assaying cell cycle checkpoints: activity of the protein kinase Chk1.

Eukaryotic cells regulate progression through the cell cycle in response to DNA damage. Cell cycle checkpoints are the signal transduction pathways that couple the detection of DNA damage to the proteins that control transitions in the cell cycle. The protein kinase Chk1, originally discovered in fission yeast, but conserved in humans, is essential for preventing mitotic entry in the presence of DNA damage or blocks to DNA replication that cannot be reconciled.

Assaying the DNA damage checkpoint in fission yeast.

Cell cycle checkpoints exist to ensure the proper maintenance and stable inheritance of genomic information. The pathways that insure the faithful execution of these checkpoints are well conserved throughout evolution. In the fission yeast, Schizosaccharomyces pombe, a major cell cycle checkpoint exists that responds to the presence of damaged DNA and prevents this damage from being propagated to future generations.

A novel protein with similarities to Rb binding protein 2 compensates for loss of Chk1 function and affects histone modification in fission yeast.

The conserved protein kinase Chk1 mediates cell cycle progression and consequently the ability of cells to survive when exposed to DNA damaging agents. Cells deficient in Chk1 are hypersensitive to such agents and enter mitosis in the presence of damaged DNA, whereas checkpoint-proficient cells delay mitotic entry to permit time for DNA repair. In a search for proteins that can improve the survival of Chk1-deficient cells exposed to DNA damage, we identified fission yeast Msc1, which is homologous to a mammalian protein that binds to the tumor suppressor Rb (RBP2).

Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest.

In the fission yeast Schizosaccharomyces pombe, the protein kinase Chk1 has an essential role in transducing a delay signal to the cell cycle machinery in the presence of DNA damage. Fission yeast cells lacking the chk1 gene do not delay progression of the cell cycle in response to damage and are thus sensitive to DNA damaging agents. We have previously shown that Chk1 is phosphorylated following DNA damage induced by a variety of agents and that this is dependent on the integrity of the DNA damage checkpoint pathway, including Rad3, the ATR homolog.

The Ded1 DEAD box helicase interacts with Chk1 and Cdc2.

Ded1 is a fission yeast DEAD box protein involved in translation. We isolated Ded1 in a screen for multi-copy suppressors of a cold-sensitive, loss-of-function mutant of the cyclin-dependent kinase Cdc2. The checkpoint protein kinase Chk1, required for cell cycle arrest in response to DNA damage, was also isolated in this screen.