Isolation and characterization of the Schizosaccharomyces pombe rhp9 gene: a gene required for the DNA damage checkpoint but not the replication checkpoint.

Checkpoint controls exist in eukaryotic cells to ensure that cells do not enter mitosis in the presence of DNA damage or unreplicated chromosomes. In Schizosaccharomyces pombe many of the checkpoint genes analysed to date are required for both the DNA damage and the replication checkpoints, an exception being chk1 . We report here on the characterization of nine new methylmethane sulphonate (MMS)-sensitive S.pombe mutants, one of which is defective in the DNA damage checkpoint but not the replication checkpoint. We have cloned and sequenced the corresponding gene. The predicted protein is most similar to the Saccharomyces cerevisiae Rad9 protein, having 46% similarity and 26% identity. The S.pombe protein, which we have named Rhp9 (Rad9 homologue in S. pombe) on the basis of structural and phenotypic similarity, also contains motifs present in BRCA1 and 53BP1. Deletion of the gene is not lethal and results in a DNA damage checkpoint defect. Epistasis analysis with other S.pombe checkpoint mutants indicates that rhp9 acts in a process involving the checkpoint rad genes and that the rhp9 mutant is phenotypically very similar to chk1.     

Role of exonucleolytic degradation in group I intron homing in phage T4

The Schizosaccharomyces pombe checkpoint gene named rad3(+) encodes an ATM-homologous protein kinase that shares a highly conserved motif with proteins involved in DNA metabolism. Previous studies have shown that Rad3 fulfills its function via the regulation of the Chk1 and Cds1 protein kinases. Here we describe a novel role for Rad3 in the control of telomere integrity. Mutations in the rad3(+) gene alleviated telomeric silencing and produced shortened lengths in the telomere repeat tracts. Genetic analysis revealed that the other checkpoint rad mutations rad1, rad17, and rad26 belong to the same phenotypic class with rad3 with regard to control of the telomere length. Of these mutations, rad3 and rad26 have a drastic effect on telomere shortening. tel1(+), another ATM homologue in S. pombe, carries out its telomere maintenance function in parallel with the checkpoint rad genes. Furthermore, either a single or double disruption of cds1(+) and chk1(+) caused no obvious changes in the telomeric DNA structure. Our results demonstrate a novel role of the S. pombe ATM homologues that is independent of chk1(+) and cds1(+).     

DNA replication checkpoint control mediated by the spindle checkpoint protein Mad2p in fission yeast

The relationship between the DNA replication and spindle checkpoints of the cell cycle is unclear, given that in most eukaryotes, spindle formation occurs only after DNA replication is complete. Fission yeast rad3 mutant cells, which are deficient in DNA replication checkpoint function, enter, progress through, and exit mitosis even when DNA replication is blocked. In contrast, the entry of cds1 mutant cells into mitosis is delayed by several hours when DNA replication is inhibited. We show here that this delay in mitotic entry in cds1 cells is due in part to activation of the spindle checkpoint protein Mad2p. In the presence of the DNA replication inhibitor hydroxyurea (HU), cds1 mad2 cells entered and progressed through mitosis earlier than did cds1 cells. Overexpression of Mad2p or inactivation of Slp1p, a regulator of the anaphase-promoting complex, also rescued the checkpoint defect of HU-treated rad3 cells. Rad3p was shown to be involved in the physical interaction between Mad2p and Slp1p in the presence of HU. These results suggested that Mad2p and Slp1p act downstream of Rad3p in the DNA replication checkpoint and that Mad2p is required for the DNA replication checkpoint when Cds1p is compromised.     

DNA polymerase δ is required for the replication feedback control of cell cycle progression in Schizosaccharomyces pombe

DNA replication and DNA repair are essential cell cycle steps ensuring correct transmission of the genome. The feedback replication control system links mitosis to completion of DNA replication and partially overlaps the radiation checkpoint control. Deletion of the chkl/rad27 gene abolishes the radiation but not the replication feedback control. Thermosensitive mutations in the DNA polymerase λ, cdc18 or cdc20 genes lead cells to arrest in the S phase of the cell cycle. We show that strains carrying any of these mutations enter lethal mitosis in the absence of the radiation checkpoint chk1/rad27. We interpret these data as an indication that an assembled replisome is essential for replication dependent control of mitosis and we propose that the arrest of the cell cycle in the thermosensitive mutants is due to the chk1 ⁺/rad27 ⁺ pathway, which monitors directly DNA for signs of damage.     

Regulation of Cdc2p and Cdc13p is required for cell cycle arrest induced by defective RNA splicing in fission yeast

Screening of cdc mutants of fission yeast for those whose cell cycle arrest is independent of the DNA damage checkpoint identified the RNA splicing-deficient cdc28 mutant. A search for mutants of cdc28 cells that enter mitosis with unspliced RNA resulted in the identification of an orb5 point mutant. The orb5+ gene, which encodes a catalytic subunit of casein kinase II, was found to be required for cell cycle arrest in other mutants with defective RNA metabolism but not for operation of the DNA replication or DNA damage checkpoints. Loss of function of wee1+ or rad24+ also suppressed the arrest of several splicing mutants. Overexpression of the major B-type cyclin Cdc13p induced cdc28 cells to enter mitosis. The abundance of Cdc13p was reduced, and the phosphorylation of Cdc2p on tyrosine 15 was maintained in splicing-defective cells. These results suggest that regulation of Cdc13p and Cdc2p is required for G2 arrest in splicing mutants.     

DNA Damage and Replication Checkpoints in Fission Yeast Require Nuclear Exclusion of the Cdc25 Phosphatase via 14-3-3 Binding

In fission yeast as well as in higher eukaryotic organisms, entry into mitosis is delayed in cells containing damaged or unreplicated DNA. This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins. In this study, we generated a mutant of fission yeast Cdc25 that is severely impaired in its ability to bind 14-3-3 proteins. Loss of both the DNA damage and replication checkpoints was observed in fission yeast cells expressing the 14-3-3 binding mutant. These findings indicate that 14-3-3 binding to Cdc25 is required for fission yeast cells to arrest their cell cycle in response to DNA damage and replication blocks. Furthermore, the 14-3-3 binding mutant localized almost exclusively to the nucleus, unlike wild-type Cdc25, which localized to both the cytoplasm and the nucleus. Nuclear accumulation of wild-type Cdc25 was observed when fission yeast cells were treated with leptomycin B, indicating that Cdc25 is actively exported from the nucleus. Nuclear exclusion of wild-type Cdc25 was observed upon overproduction of Rad 24, one of the two fission yeast 14-3-3 proteins, indicating that one function of Rad 24 is to keep Cdc25 out of the nucleus. In support of this conclusion, Rad 24 overproduction did not alter the nuclear location of the 14-3-3 binding mutant. These results indicate that 14-3-3 binding contributes to the nuclear exclusion of Cdc25 and that the nuclear exclusion of Cdc25 is required for a normal checkpoint response to both damaged and unreplicated DNA.     

The DNA damage checkpoint and PKA pathways converge on APC substrates and Cdc20 to regulate mitotic progression

The conserved checkpoint kinases Chk1 and Rad53-Dun1 block the metaphase to anaphase transition by the phosphorylation and stabilization of securin, and block the mitotic exit network regulated by the Bfa1-Bub2 complex. However, both chk1 and rad53 mutants are able to exit from mitosis and initiate a new cell cycle, suggesting that both pathways have supporting functions in restraining anaphase and in blocking the inactivation of mitotic cyclin-Cdk1 complexes. Here we find that the cyclic-AMP-dependent protein kinase (PKA) pathway supports Chk1 in the regulation of mitosis by targeting the mitotic inducer Cdc20. Cdc20 is phosphorylated on PKA consensus sites after DNA damage, and this phosphorylation requires the Atr orthologue Mec1 and the PKA catalytic subunits Tpk1 and Tpk2. We show that the inactivation of PKA or expression of phosphorylation-defective Cdc20 proteins accelerates securin and Clb2 destruction in chk1 mutants and is sufficient to remove most of the DNA damage-induced delay. Mutation of the Cdc20 phosphorylation sites permitted the interaction of Cdc20 with Clb2 under conditions that should halt cell cycle progression. These data show that PKA pathways regulate mitotic progression through Cdc20 and support the DNA damage checkpoint pathways in regulating the destruction of Clb2 and securin.     

Recovery from DNA damage checkpoint arrest by PP1-mediated inhibition of Chk1

The G2 DNA damage checkpoint delays mitotic entry via the upregulation of Wee1 kinase and the downregulation of Cdc25 phosphatase by Chk1 kinase, and resultant inhibitory phosphorylation of Cdc2. While checkpoint activation is well understood, little is known about how the checkpoint is switched off to allow cell cycle re-entry. To identify proteins required for checkpoint release, we screened for genes in Schizosaccharomyces pombe that, when overexpressed, result in precocious mitotic entry in the presence of DNA damage. We show that overexpression of the type I protein phosphatase Dis2 sensitises S. pombe cells to DNA damage, causing aberrant mitoses. Dis2 abrogates Chk1 phosphorylation and activation in vivo, and dephosphorylates Chk1 and a phospho-S345 Chk1 peptide in vitro. dis2Delta cells have a prolonged chk1-dependent arrest and a compromised ability to downregulate Chk1 activity for checkpoint release. These effects are specific for the DNA damage checkpoint, because Dis2 has no effect on the chk1-independent response to stalled replication forks. We propose that inactivation of Chk1 by Dis2 allows mitotic entry following repair of DNA damage in the G2-phase.     

Rad3 and Sty1 function in Schizosaccharomyces pombe: an integrated response to DNA damage and environmental stress?

In Schizosaccharomyces pombe , the Ataxia Telangiectasia-mutated (Atm)/Atm and Rad 3 Related (Atr) homologue Rad3 is an essential regulator of the response to DNA damage and stalled replication forks. Rad3 activates the downstream kinases Chk1 and Cds1. These kinases in turn inhibit cell cycle progression by mediating Cdc2 phosphorylation. Studies in both yeast and mammalian cells suggest additional roles for Rad3 in regulating cellular responses to environmental stress. In S. pombe , cellular responses to various environmental stresses are regulated primarily through the stress-activated MAP kinase p38 homologue Sty1. An important function of Sty1 is to drive cells rapidly through mitosis by facilitating the accumulation of Cdc25. Interestingly, Sty1 is activated simultaneously with Rad3 following exposure to UV radiation or ionizing radiation (IR). Similarly, exposure to environmental stresses induces the expression of rad3 ⁺, cds1 ⁺ and other checkpoint regulator genes. It is currently unclear how the pathways regulated by Sty1 and Rad3 and their opposing effects on mitosis are integrated. Recent studies suggest that Sty1 and Rad3 function together to regulate the expression of several stress response genes following exposure to IR. In this review, we discuss current knowledge on the interaction of Rad3/Atm and Sty1/p38 in regulating cellular responses to environmental stress and DNA damage.     

Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.

Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. Keywords: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3     

Maintenance of replication forks and the S-phase checkpoint by Cdc18p and Orp1p

S-phase and DNA damage checkpoint controls block the onset of mitosis when DNA is damaged or DNA replication is incomplete. It has been proposed that damaged or incompletely replicated DNA generates structures that are sensed by the checkpoint control pathway, although little is known about the structures and mechanisms involved. Here, we show that the DNA replication initiation proteins Orp1p and Cdc18p are required to induce and maintain the S-phase checkpoint in Schizosaccharomyces pombe. The presence of DNA replication structures correlates with activation of the Cds1p checkpoint protein kinase and the S-phase checkpoint pathway. By contrast, induction of the DNA damage pathway is not dependent on Orp1p or Cdc18p. We propose that the presence of unresolved replication forks, together with Orp1p and Cdc18p, are necessary to activate the Cds1p-dependent S-phase checkpoint.     

Control of the Cdc2/cyclin B complex in Xenopus egg extracts arrested at a G2/M checkpoint with DNA synthesis inhibitors.

Proliferating eukaryotic cells possess checkpoint mechanisms that block cell division in the presence of unreplicated or damaged DNA. Using cell-free extracts from Xenopus eggs, we have investigated the mechanisms underlying the inability of a recombinant Cdc2/cyclin B complex to induce mitosis in the presence of incompletely replicated DNA. We found that the activities of the kinases and phosphatases that regulate the major phosphorylation sites on Cdc2 (e.g., tyrosine 15, threonine 14, and threonine 161) are not altered significantly under conditions where Xenopus extracts remain stably arrested in interphase due to the presence of the replication inhibitor aphidicolin. However, at threshold concentrations, a Cdc2/cyclin B complex containing a mutant Cdc2 subunit that cannot be phosphorylated on either tyrosine 15 or threonine 14 displays a markedly reduced capacity to induce mitosis in the presence of aphidicolin. This observation indicates that the replication checkpoint in Xenopus egg extracts functions without the inhibitory tyrosine and threonine phosphorylation of Cdc2. We provide evidence that the checkpoint-dependent suppression of the Cdc2/cyclin B complex involves a titratable inhibitor that is regulated by the presence of unreplicated DNA.     

Checkpoint-dependent and -independent roles of Swi3 in replication fork recovery and sister chromatid cohesion in fission yeast

Multiple genome maintenance processes are coordinated at the replication fork to preserve genomic integrity. How eukaryotic cells accomplish such a coordination is unknown. Swi1 and Swi3 form the replication fork protection complex and are involved in various processes including stabilization of replication forks, activation of the Cds1 checkpoint kinase and establishment of sister chromatid cohesion in fission yeast. However, the mechanisms by which the Swi1-Swi3 complex achieves and coordinates these tasks are not well understood. Here, we describe the identification of separation-of-function mutants of Swi3, aimed at dissecting the molecular pathways that require Swi1-Swi3. Unlike swi3 deletion mutants, the separation-of-function mutants were not sensitive to agents that stall replication forks. However, they were highly sensitive to camptothecin that induces replication fork breakage. In addition, these mutants were defective in replication fork regeneration and sister chromatid cohesion. Interestingly, unlike swi3-deleted cell, the separation-of-functions mutants were proficient in the activation of the replication checkpoint, but their fork regeneration defects were more severe than those of checkpoint mutants including cds1Δ, chk1Δ and rad3Δ. These results suggest that, while Swi3 mediates full activation of the replication checkpoint in response to stalled replication forks, Swi3 activates a checkpoint-independent pathway to facilitate recovery of collapsed replication forks and the establishment of sister chromatid cohesion. Thus, our separation-of-function alleles provide new insight into understanding the multiple roles of Swi1-Swi3 in fork protection during DNA replication, and into understanding how replication forks are maintained in response to different genotoxic agents.     

Cdc6 and DNA replication: Limited to humble origins

The budding yeast Cdc6 protein is important for regulating DNA replication intiation. Cdc6p acts at replication origins, and cdc6-1 mutants arrest with unreplicated DNA and show elevated minichromosome loss rates. Overexpression of the related Cdc 18 protein in fission yeast results in DNA rereplication; however, Cdc6p overexpression does not cause this result. A recent paper⁽¹⁾ further defines the role of Cdc6p in DNA replication. Cdc6p only promotes DNA replication between the end of mitosis and late G₁, and although the Cdc6 protein is highly unstable, neither degradation nor nuclear localization is critical for limiting DNA replication to this interval.     

Chl12 (Ctf18) forms a novel replication factor C-related complex and functions redundantly with Rad24 in the DNA replication checkpoint pathway

RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. The rad24Delta mutation enhances the defect of rfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint. CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that although neither chl12Delta nor rad24Delta single mutants are defective, chl12Delta rad24Delta double mutants become defective in the replication block checkpoint. We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes. Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint.     

Fission yeast rad17: a homologue of budding yeast RAD24 that shares regions of sequence similarity with DNA polymerase accessory proteins.

Following DNA damage or a block to DNA synthesis, checkpoint pathways act to arrest mitosis and prevent the attempted segregation of damaged or unreplicated DNA. The rad17 locus of Schizosaccharomyces pombe is one of seven known radiation-sensitive (rad) loci which are absolutely required to prevent mitosis following DNA damage in fission yeast. Six of these (rad1, rad3, rad9, rad17, rad26 and hus1) are also required for the checkpoint which prevents mitosis from occurring before DNA replication is complete. We report here that the predicted rad17 gene product is a basic hydrophilic protein of 606 amino acids which contains five domains with sequence homology to replication factor C (RF-C)/activator 1 subunits. Western analysis and fusion with Green Fluorescent Protein indicate that the abundance and electrophoretic mobility of Rad17 is not significantly modified following a block to DNA synthesis or following DNA damage, and that Rad17 is localized in the nucleus. Rad17 function is not essential for growth, but is required for the function of the DNA structure-dependent checkpoints. Site-directed mutagenesis has been used to demonstrate the biological significance of the RF-C/activator 1-related domains. These studies have also defined an element of the radiation sensitivity caused by loss of Rad17 function which is not associated with the radiation-induced G2 arrest defect seen in the rad17.d null mutant cells.     

p56(chk1) protein kinase is required for the DNA replication checkpoint at 37 degrees C in fission yeast.

Fission yeast p56(chk1) kinase is known to be involved in the DNA damage checkpoint but not to be required for cell cycle arrest following exposure to the DNA replication inhibitor hydroxyurea (HU). For this reason, p56(chk1) is considered not to be necessary for the DNA replication checkpoint which acts through the inhibitory phosphorylation of p34(cdc2) kinase activity. In a search for Schizosaccharomyces pombe mutants that abolish the S phase cell cycle arrest of a thermosensitive DNA polymerase delta strain at 37 degrees C, we isolated two chk1 alleles. These alleles are proficient for the DNA damage checkpoint, but induce mitotic catastrophe in several S phase thermosensitive mutants. We show that the mitotic catastrophe correlates with a decreased level of tyrosine phosphorylation of p34(cdc2). In addition, we found that the deletion of chk1 and the chk1 alleles abolish the cell cycle arrest and induce mitotic catastrophe in cells exposed to HU, if the cells are grown at 37 degrees C. These findings suggest that chk1 is important for the maintenance of the DNA replication checkpoint in S phase thermosensitive mutants and that the p56(chk1) kinase must possess a novel function that prevents premature activation of p34(cdc2) kinase under conditions of impaired DNA replication at 37 degrees C.     

The role of Cdc25 phosphatases in cell cycle checkpoints

Summary The major driving forces in the eukaryotic cell cycle are the cyclin-dependent kinases (Cdk). Cdks can be activated through dephosphorylation of inhibitory phosphorylations catalyzed by the Cdc25 phosphatase family. In higher-eukaryotic cells, there exist three Cdc25 family members, Cdc25A, Cdc25B, and Cdc25C. While Cdc25A plays a major role at the G₁-to-S phase transition, Cdc25B and C are required for entry into mitosis. The regulation of Cdc25C is crucial for the operation of the DNA-damage checkpoint. Two protein kinases, Chk1 and Cds1, can be activated in response to DNA damage or in the presence of unreplicated DNA. Chk1 and Cds1 may phosphorylate Cdc25C to prevent entry into mitosis through inhibition of Cdc2 (Cdk1) dephosphorylation.