Mechanism of caffeine-induced checkpoint override in fission yeast

Mitotic checkpoints restrain the onset of mitosis (M) when DNA is incompletely replicated or damaged. These checkpoints are conserved between the fission yeast Schizosaccharomyces pombe and mammals. In both types of organisms, the methylxanthine caffeine overrides the synthesis (S)-M checkpoint that couples mitosis to completion of DNA S phase. The molecular target of caffeine was sought in fission yeast. Caffeine prevented activation of Cds1 and phosphorylation of Chk1, two protein kinases that enforce the S-M checkpoint triggered by hydroxyurea. Caffeine did not inhibit these kinases in vitro but did inhibit Rad3, a kinase that regulates Cds1 and Chk1. In accordance with this finding, caffeine also overrode the G(2)-M DNA damage checkpoint that requires Rad3 function. Rad3 coprecipitated with Cds1 expressed at endogenous amounts, a finding that supports the hypothesis that Rad3 is involved in direct activation of Cds1.     

Replication checkpoint kinase Cds1 regulates recombinational repair protein Rad60

Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks. How Cds1 accomplishes this task is unknown. We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast. Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization. A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest. Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair. These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1. We propose that control of Rad60 regulates recombination events at stalled forks.     

G(1)/S CDK is inhibited to restrain mitotic onset when DNA replication is blocked in fission yeast

Cyclin-dependent kinase (CDK) Tyr15 phosphorylation plays a major role in regulating G(2)/M CDKs, but the role of this phosphorylation in regulating G(1)/S CDKs is less clear. We have studied the regulation and function of Cdc2-Tyr15 phosphorylation in the fission yeast Schizosaccharomyces pombe G(1)/S CDK Cig2/Cdc2. This complex is subject to high level Cdc2-Tyr15 phosphorylation inhibiting its kinase activity in hydroxyurea-treated cells blocked in S-phase. We show that this Tyr15 phosphorylation is required to maintain efficient mitotic checkpoint arrest, because Cig2 accumulates during the block and this accumulation can advance mitotic onset. This mitotic induction operates, at least in part, through activation of the normal G(2)/M CDK complex Cdc13/Cdc2. Thus, Tyr15 phosphorylation of G(1)/S CDK complexes is important in the checkpoint control blocking mitotic onset when DNA replication is inhibited.     

The checkpoint-dependent nuclear accumulation of Rho1p exchange factor Rgf1p is important for tolerance to chronic replication stress

Guanine nucleotide exchange factors control many aspects of cell morphogenesis by turning on Rho-GTPases. The fission yeast exchange factor Rgf1p (Rho gef1) specifically regulates Rho1p during polarized growth and localizes to cortical sites. Here we report that Rgf1p is relocalized to the cell nucleus during the stalled replication caused by hydroxyurea (HU). Import to the nucleus is mediated by a nuclear localization sequence at the N-terminus of Rgf1p, whereas release into the cytoplasm requires two leucine-rich nuclear export sequences at the C-terminus. Moreover, Rgf1p nuclear accumulation during replication arrest depends on the 14-3-3 chaperone Rad24p and the DNA replication checkpoint kinase Cds1p. Both proteins control the nuclear accumulation of Rgf1p by inhibition of its nuclear export. A mutant, Rgf1p-9A, that substitutes nine serine potential phosphorylation Cds1p sites for alanine fails to accumulate in the nucleus in response to replication stress, and this correlates with a severe defect in survival in the presence of HU. In conclusion, we propose that the regulation of Rgf1p could be part of the mechanism by which Cds1p and Rad24p promote survival in the presence of chronic replication stress. It will be of general interest to understand whether the same is true for homologues of Rgf1p in budding yeast and higher eukaryotes.     

The phosphorylation network for efficient activation of the DNA replication checkpoint in fission yeast

Protein phosphorylation is the hallmark of checkpoint activation. Hundreds of targets of checkpoint kinases have been identified recently by genome-wide investigations. However, the complete picture of a phosphorylation network required for activation of a checkpoint pathway has not been available. The DNA replication checkpoint in Schizosaccharomyces pombe contains two major protein kinases, the sensor kinase Rad3 and the effector kinase Cds1, with the latter mediating most of the checkpoint functions. We show here that when DNA replication is arrested, efficient activation of Cds1 requires five phosphorylations that cooperate in a parallel or a sequential manner. Phosphorylation of a threonine residue (Thr(11)) in Cds1 by Rad3 occurs at a basal level in the absence of three other parallel Rad3-dependent phosphorylations on the mediator Mrc1 and Rad9 in the checkpoint clamp complex. However, the three parallel Rad3-dependent phosphorylations are all required for efficient phosphorylation of Thr(11) in Cds1 by Rad3. Phosphorylation of Thr(11) has been shown previously to promote autophosphorylation of Thr(328) in the kinase domain of Cds1, which directly activates the enzyme, leading to full activation of the checkpoint pathway. Interestingly, phosphorylation of Mrc1 by Rad3 does not require the phosphorylation of Rad9, suggesting that activation of the sensor kinase Rad3 in the replication checkpoint of fission yeast may involve a different mechanism.     

Cds1 phosphorylation by Rad3-Rad26 kinase is mediated by forkhead-associated domain interaction with Mrc1

The protein kinase Cds1 is an effector of the replication checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required to stabilize stalled replication forks, and it helps to prevent the onset of mitosis until the genome is fully replicated. Mrc1 (mediator of the replication checkpoint-1) and Rad3-Rad26 kinase are required for Cds1 activation, but exactly how Mrc1 mediates Cds1 activation is unknown. Here we show that Mrc1 is required for the initial threonine 11 phosphorylation of Cds1 by Rad3-Rad26. Mrc1 specifically interacts with the forkhead-associated (FHA) domain of Cds1 in yeast two-hybrid assays. Mutations in the FHA domain that abolish this interaction also eliminate Thr-11 phosphorylation of Cds1. Weak Thr-11 phosphorylation of a "kinase-dead" mutant of Cds1 is rescued by co-expression of wild type Cds1. The requirement for Mrc1 in the replication checkpoint can be partially eliminated by expression of a Rad26-Cds1 fusion protein. These findings suggest that recognition of Mrc1 by the FHA domain of Cds1 serves to recruit Cds1 to Rad3-Rad26. This interaction mediates the initial Thr-11 phosphorylation of Cds1 by Rad3-Rad26 with subsequent intermolecular phosphorylation events leading to full activation of Cds1.     

Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote s-phase genome stability

The minichromosome maintenance (MCM) complex plays essential, conserved roles throughout DNA synthesis: first, as a component of the prereplication complex at origins and, then, as a helicase associated with replication forks. Here we use fission yeast (Schizosaccharomyces pombe) as a model to demonstrate a role for the MCM complex in protecting replication fork structure and promoting recovery from replication arrest. Loss of MCM function generates lethal double-strand breaks at sites of DNA synthesis during replication elongation, suggesting replication fork collapse. MCM function also maintains the stability of forks stalled by hydroxyurea that activate the replication checkpoint. In cells where the checkpoint is activated, Mcm4 binds the Cds1 kinase and undergoes Cds1-dependent phosphorylation. MCM proteins also interact with proteins involved in homologous recombination, which promotes recovery from arrest by ensuring normal mitosis. We suggest that the MCM complex links replication fork stabilization with checkpoint arrest and recovery through direct interactions with checkpoint and recombination proteins and that this role in S-phase genome stability is conserved from yeast to human cells.     

Tel2 is required for activation of the Mrc1-mediated replication checkpoint

Proteins belonging to the Tel2/Rad-5/Clk-2 family are conserved among eukaryotes and are involved in various cellular processes, such as cell proliferation, telomere maintenance, the biological clock, and the DNA damage checkpoint. However, the molecular mechanisms underlying the functions of these molecules remain largely unclear. Here we report that in the fission yeast, Schizosaccharomyces pombe, Tel2 is required for efficient phosphorylation of Mrc1, a mediator of DNA replication checkpoint signaling, and for activation of Cds1, a replication checkpoint kinase, when DNA replication is blocked by hydroxyurea. In fact, Tel2 is required for survival of replication fork arrest and for the replication checkpoint in cells lacking Chk1, another checkpoint kinase the role of which overlaps that of Cds1 in cell cycle arrest by replication block. In addition, Tel2 plays important roles in entry into S phase and in genome stability. Tel2 is essential for vegetative cell growth, and the tel2Delta strain accumulated cells with 1C DNA content after germination. In the absence of hydroxyurea, Tel2 is vital in the mutant lacking Swi1, a component of the replication fork protection complex, and multiple Rad22 DNA repair foci were frequently observed in Tel2-repressed swi1Delta cells especially at S phase. In contrast, the cds1Deltaswi1Delta mutant did not show such lethality. These results indicate that S. pombe Tel2 plays important roles in the Mrc1-mediated replication checkpoint as well as in the Cds1-independent regulation of genome integrity.     

Basis for the Checkpoint Signal Specificity That Regulates Chk1 and Cds1 Protein Kinases

Six checkpoint Rad proteins (Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1) are needed to regulate checkpoint protein kinases Chk1 and Cds1 in fission yeast. Chk1 is required to prevent mitosis when DNA is damaged by ionizing radiation (IR), whereas either kinase is sufficient to prevent mitosis when DNA replication is inhibited by hydroxyurea (HU). Checkpoint Rad proteins are required for IR-induced phosphorylation of Chk1 and HU-induced activation of Cds1. IR activates Cds1 only during the DNA synthesis (S) phase, whereas HU induces Chk1 phosphorylation only in cds1 mutants. Here, we investigate the basis of the checkpoint signal specificity of Chk1 phosphorylation and Cds1 activation. We show that IR fails to induce Chk1 phosphorylation in HU-arrested cells. Release from the HU arrest following IR causes substantial Chk1 phosphorylation. These and other data indicate that Cds1 prevents Chk1 phosphorylation in HU-arrested cells, which suggests that Cds1 actively suppresses a repair process that leads to Chk1 phosphorylation. Cds1 becomes more highly concentrated in the nucleus only during the S phase of the cell cycle. This finding correlates with S-phase specificity of IR-induced activation of Cds1. However, constitutive nuclear localization of Cds1 does not enhance IR-induced activation of Cds1. This result suggests that Cds1 activation requires DNA structures or protein activities that are present only during S phase. These findings help to explain how Chk1 and Cds1 respond to different checkpoint signals.     

Replication checkpoint protein Mrc1 is regulated by Rad3 and Tel1 in fission yeast

Fission yeast Mrc1 (mediator of replication checkpoint 1) is an adaptor checkpoint protein required for Rad3-dependent activation of the checkpoint kinase Cds1 in response to arrest of replication forks. Here we report studies on the regulation of Mrc1 by phosphorylation. Replication arrest induced by hydroxyurea (HU) induces Mrc1 phosphorylation that is detected by a change in Mrc1 electrophoretic mobility. Phosphorylation is maintained in cds1Delta, rad3Delta, and tel1Delta single mutants but eliminated in a rad3Delta tel1Delta double mutant. Mrc1 has two clusters of S/TQ motifs that are potential Rad3/Tel1 phosphorylation sites. Mutation of six S/TQ motifs in these two clusters strongly impairs Mrc1 phosphorylation. Two motifs located at S604 and T645 are vital for HU resistance. The T645A mutation strongly impairs a Cds1-Mrc1 yeast two-hybrid interaction that is dependent on a functional forkhead-associated (FHA) domain in Cds1, indicating that phosphorylation of T645 mediates Mrc1's association with Cds1. Consistent with this model, the T645 region of Mrc1 effectively substitutes for the T11 region of Cds1 that is thought to be phosphorylated by Rad3 and to mediate FHA-dependent oligomerization of Cds1. The S/TQ cluster that includes S604 is needed for Mrc1's increased association with chromatin in replication-arrested cells. These data indicate that Rad3 and Tel1 regulate Mrc1 through differential phosphorylation to control Cds1.