The fission yeast S-phase cyclin Cig2 can drive mitosis

Commitment to mitosis is regulated by cyclin-dependent kinase (CDK) activity. In the fission yeast Schizosaccharomyces pombe, the major B-type cyclin, Cdc13, is necessary and sufficient to drive mitotic entry. Furthermore, Cdc13 is also sufficient to drive S phase, demonstrating that a single cyclin can regulate alternating rounds of replication and mitosis, and providing the foundation of the quantitative model of CDK function. It has been assumed that Cig2, a B-type cyclin expressed only during S phase and incapable of driving mitosis in wild-type cells, was specialized for S-phase regulation. Here, we show that Cig2 is capable of driving mitosis. Cig2/CDK activity drives mitotic catastrophe—lethal mitosis in inviably small cells—in cells that lack CDK inhibition by tyrosine-phosphorylation. Moreover, Cig2/CDK can drive mitosis in the absence of Cdc13/CDK activity and constitutive expression of Cig2 can rescue loss of Cdc13 activity. These results demonstrate that in fission yeast, not only can the presumptive M-phase cyclin drive S phase, but the presumptive S-phase cyclin can drive M phase, further supporting the quantitative model of CDK function. Furthermore, these results provide an explanation, previously proposed on the basis of computational analyses, for the surprising observation that cells expressing a single-chain Cdc13-Cdc2 CDK do not require Y15 phosphorylation for viability. Their viability is due to the fact that in such cells, which lack Cig2/CDK complexes, Cdc13/CDK activity is unable to drive mitotic catastrophe.     

S-phase checkpoint controls mitosis via an APC-independent Cdc20p function

Cells divide with remarkable fidelity, allowing complex organisms to develop and possess longevity. Checkpoint controls contribute by ensuring that genome duplication and segregation occur without error so that genomic instability, associated with developmental abnormalities and a hallmark of most human cancers, is avoided. S-phase checkpoints prevent cell division while DNA is replicating. Budding yeast Mec1p and Rad53p, homologues of human checkpoint kinases ATM/ATR and Chk2, are needed for this control system. How Mec1p and Rad53p prevent mitosis in S phase is not known. Here we provide evidence that budding yeasts avoid mitosis during S phase by regulating the anaphase-promoting complex (APC) specificity factor Cdc20p: Mec1p and Rad53p repress the accumulation of Cdc20p in S phase. Because precocious Cdc20p accumulation causes anaphase onset and aneuploidy, Cdc20p concentrations must be precisely regulated during each and every cell cycle. Catastrophic mitosis induced by Cdc20p in S phase occurs even in the absence of core APC components. Thus, Cdc20p can function independently of the APC.     

Multiple functions of the S-phase checkpoint mediator

There is mounting evidence that replication defects are the major source of spontaneous genomic instability in cells, and that S-phase checkpoints are the principal defense against such instability. The S-phase checkpoint mediator protein Mrc1/Claspin mediates the checkpoint response to replication stress by facilitating phosphorylation of effector kinase by a sensor kinase. In this review, the multiple functions and the regulation of the S-phase checkpoint mediator are discussed.     

Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.     

Recruitment of the cell cycle checkpoint kinase ATR to chromatin during S-phase

The ataxia telangiectasia-mutated (ATM) and Rad3-related kinase (ATR) is a central component of the cell cycle checkpoint machinery required to induce cell cycle arrest in response to DNA damage. Accumulating evidence suggests a role for ATR in signaling DNA damage during S-phase. Here we show that ATR is recruited to nuclear foci induced by replication fork stalling in a manner that is dependent on the single stranded binding protein replication protein A (RPA). ATR associates with chromatin in asynchronous cell cultures, and we use a variety of approaches to examine the association of ATR with chromatin in the absence of agents that cause genotoxic stress. Under our experimental conditions, ATR exhibits a decreased affinity for chromatin in quiescent cells and cells synchronized at mitosis but an increased affinity for chromatin as cells re-enter the cell cycle. Using centrifugal elutriation to obtain cells enriched at various stages of the cell cycle, we show that ATR associates with chromatin in a cell cycle-dependent manner, specifically during S-phase. Cell cycle association of ATR with chromatin mirrors that of RPA in addition to claspin, a cell cycle checkpoint protein previously shown to be a component of the replication machinery. Furthermore, association of ATR with chromatin occurs in the absence of detectable DNA damage and cell cycle checkpoint activation. These data are consistent with a model whereby ATR is recruited to chromatin during the unperturbed cell cycle and points to a role of ATR in monitoring genome integrity during normal S-phase progression.     

Chromatin dynamics from S-phase to mitosis: contributions of histone modifications

This review focuses on the major protein moiety of chromosomes, i.e., the histone proteins, on the contribution of their posttranslational modification to structural and functional chromatin dynamics, on the acetylation and methylation of lysine residues, and on the phosphorylation of serine or threonine with respect to various steps during the cell cycle.     

The S-phase checkpoint and its regulation in Saccharomyces cerevisiae

Cells are never more vulnerable than during DNA replication, which represents a major moment of potential genetic instability. Genotoxic insults induce many different forms of DNA damage that may interfere with the ability of cells to properly duplicate their genome. Primary damage may in turn undergo structural transformations during DNA replication, thus generating secondary lesions that may be even more dangerous. Cells experiencing replication of damaged DNA or replication blocks activate an S-phase checkpoint response that assures the fidelity and completion of DNA replication before cells enter M-phase. The S-phase checkpoint pathway regulates not only progress through the cell cycle but also DNA repair and DNA replication itself.     

Testing cyclin specificity in the exit from mitosis

Cyclical inactivation of B-type cyclins has been proposed to be required for alternating DNA replication and mitosis. Destruction box-dependent Clb5p degradation is strongly increased in mitotic cells, and constitutive overexpression of Clb5p lacking the destruction box resulted in rapid accumulation of inviable cells, frequently multiply budded, with DNA contents ranging from unreplicated to apparently fully replicated. Loss of viability correlated with retention of nuclear Clb5p at the time of nuclear division. CLB2-Deltadb overexpression that was quantitatively comparable to CLB5-Deltadb overexpression with respect to Clb protein production and Clb-associated kinase activity resulted in a distinct phenotype: reversible mitotic arrest with uniformly replicated DNA. Simultaneous overexpression of CLB2-Deltadb and CLB5-Deltadb overexpressers similarly resulted in a uniform arrest with replicated DNA, and this arrest was significantly more reversible than that observed with CLB5-Deltadb overexpression alone. These results suggest that Clb2p and not Clb5p can efficiently block mitotic completion. We speculate that CLB5-Deltadb overexpression may be lethal, because persistence of high nuclear Clb5p-associated kinase throughout mitosis leads to failure to load origins of replication, thus preventing DNA replication in the succeeding cell cycle.     

Regulation of DNA replication by the S-phase DNA damage checkpoint

Cells slow replication in response to DNA damage. This slowing was the first DNA damage checkpoint response discovered and its study led to the discovery of the central checkpoint kinase, Ataxia Telangiectasia Mutated (ATM). Nonetheless, the manner by which the S-phase DNA damage checkpoint slows replication is still unclear. The checkpoint could slow bulk replication by inhibiting replication origin firing or slowing replication fork progression, and both mechanisms appear to be used. However, assays in various systems using different DNA damaging agents have produced conflicting results as to the relative importance of the two mechanisms. Furthermore, although progress has been made in elucidating the mechanism of origin regulation in vertebrates, the mechanism by which forks are slowed remains unknown. We review both past and present efforts towards determining how cells slow replication in response to damage and try to resolve apparent conflicts and discrepancies within the field. We propose that inhibition of origin firing is a global checkpoint mechanism that reduces overall DNA synthesis whenever the checkpoint is activated, whereas slowing of fork progression reflects a local checkpoint mechanism that only affects replisomes as they encounter DNA damage and therefore only affects overall replication rates in cases of high lesion density.     

RPA2 is a direct downstream target for ATR to regulate the S-phase checkpoint

Upon DNA damage, replication is inhibited by the S-phase checkpoint. ATR (ataxia telangiectasia mutated- and Rad3-related) is specifically involved in the inhibition of replicon initiation when cells are treated with DNA damage-inducing agents that stall replication forks, but the mechanism by which it acts to prevent replication is not yet fully understood. We observed that RPA2 is phosphorylated on chromatin in an ATR-dependent manner when replication forks are stalled. Mutation of the ATR-dependent phosphorylation sites in RPA2 leads to a defect in the down-regulation of DNA synthesis following treatment with UV radiation, although ATR activation is not affected. Threonine 21 and serine 33, two residues among several phosphorylation sites in the amino terminus of RPA2, are specifically required for the UV-induced, ATR-mediated inhibition of DNA replication. RPA2 mutant alleles containing phospho-mimetic mutations at ATR-dependent phosphorylation sites have an impaired ability to associate with replication centers, indicating that ATR phosphorylation of RPA2 directly affects the replication function of RPA. Our studies suggest that in response to UV-induced DNA damage, ATR rapidly phosphorylates RPA2, disrupting its association with replication centers in the S-phase and contributing to the inhibition of DNA replication.     

Coordination of DNA synthesis and replicative unwinding by the S-phase checkpoint pathways

The process of DNA replication includes duplex unwinding, followed immediately by DNA synthesis. In eukaryotes, DNA synthesis is disturbed in damaged DNA regions, in replication slow zones, or as a result of insufficient nucleotide level. This review aims to discuss the mechanisms that coordinate DNA unwinding and synthesis, allowing replication to be completed even in the presence of genomic insults. There is a growing body of evidence which suggests that S-phase checkpoint pathways regulate both replicative unwinding and DNA synthesis, to synchronize the two processes, thus ensuring genome stability.     

Redundancy, insult-specific sensors and thresholds: unlocking the S-phase checkpoint response

DNA damage that is not properly repaired during genomic replication is a major source of gross chromosomal rearrangements and sequence loss during cell proliferation. In higher eukaryotes such mutations increase the risk of cancer. Eukaryotic cells have multiple checkpoint responses activated by DNA damage and stalled replication forks. We focus here on fork-associated events that activate and respond to S-phase checkpoint kinases.     

A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins.

Deletion of the fission yeast mitotic B-type cyclin gene cdc13 causes cells to undergo successive rounds of DNA replication. We have used a strain which expresses cdc13 conditionally to investigate re-replication. Activity of Start genes cdc2 and cdc10 is necessary and p34cdc2 kinase is active in re-replicating cells. We tested to see whether other cyclins were required for re-replication using cdc13delta. Further deletion of cig1 and puc1 had no effect, but deletion of cig2/cyc17 caused a severe delay in re-replication. Deletion of cig1 and cig2/cyc17 together abolished re-replication completely and cells arrested in G1. This, and analysis of the temperature sensitive cdc13-117 mutant, suggests that cdc13 can effectively substitute for the G1 cyclin activity of cig2/cyc17. We have characterized p56cdc13 activity and find evidence that in the absence of G1 cyclins, S-phase is delayed until the mitotic p34cdc2-p56cdc13 kinase is sufficiently active. These data suggest that a single oscillation of p34cdc2 kinase activity provided by a single B-type cyclin can promote ordered progression into both DNA replication and mitosis, and that the level of cyclin-dependent kinase activity may act as a master regulator dictating whether cells undergo S-phase or mitosis.     

How cyclin A destruction escapes the spindle assembly checkpoint

The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can outcompete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A–Cdc20 to the APC/C.     

Surviving chromosome replication: the many roles of the S-phase checkpoint pathway

Checkpoints were originally identified as signalling pathways that delay mitosis in response to DNA damage or defects in chromosome replication, allowing time for DNA repair to occur. The ATR (ataxia- and rad-related) and ATM (ataxia-mutated) protein kinases are recruited to defective replication forks or to sites of DNA damage, and are thought to initiate the DNA damage response in all eukaryotes. In addition to delaying cell cycle progression, however, the S-phase checkpoint pathway also controls chromosome replication and DNA repair pathways in a highly complex fashion, in order to preserve genome integrity. Much of our understanding of this regulation has come from studies of yeasts, in which the best-characterized targets are the stimulation of ribonucleotide reductase activity by multiple mechanisms, and the inhibition of new initiation events at later origins of DNA replication. In addition, however, the S-phase checkpoint also plays a more enigmatic and apparently critical role in preserving the functional integrity of defective replication forks, by mechanisms that are still understood poorly. This review considers some of the key experiments that have led to our current understanding of this highly complex pathway.     

Highly mutagenic and severely imbalanced dNTP pools can escape detection by the S-phase checkpoint

A balanced supply of deoxyribonucleoside triphosphates (dNTPs) is one of the key prerequisites for faithful genome duplication. Both the overall concentration and the balance among the individual dNTPs (dATP, dTTP, dGTP, and dCTP) are tightly regulated, primarily by the enzyme ribonucleotide reductase (RNR). We asked whether dNTP pool imbalances interfere with cell cycle progression and are detected by the S-phase checkpoint, a genome surveillance mechanism activated in response to DNA damage or replication blocks. By introducing single amino acid substitutions in loop 2 of the allosteric specificity site of Saccharomyces cerevisiae RNR, we obtained a collection of strains with various dNTP pool imbalances. Even mild dNTP pool imbalances were mutagenic, but the mutagenic potential of different dNTP pool imbalances did not directly correlate with their severity. The S-phase checkpoint was activated by the depletion of one or several dNTPs. In contrast, when none of the dNTPs was limiting for DNA replication, even extreme and mutagenic dNTP pool imbalances did not activate the S-phase checkpoint and did not interfere with the cell cycle progression.     

Novel yeast killer toxins provoke S-phase arrest and DNA damage checkpoint activation

Certain strains of Pichia acaciae and Wingea robertsiae (synonym Debaryomyces robertsiae) harbour extranuclear genetic elements that confer a killer phenotype to their host. Such killer plasmids (pPac1-2 of P. acaciae and pWR1A of W. robertsiae) were sequenced and compared with the zymocin encoding pGKL1 of Kluyveromyces lactis. Both new elements were found to be closely related to each other, but they are only partly similar to pGKL1. As for the latter, they encode functions mediating binding of the toxin to the target cell's chitin and a hydrophobic region potentially involved in uptake of a toxin subunit by target cells. Consistently, mutations affecting the target cell's major chitin synthase (Chs3) protect it from toxin action. Heterologous intracellular expression of respective open reading frames identified cell cycle-arresting toxin subunits deviating structurally from the likewise imported gamma-subunit of the K. lactis zymocin. Accordingly, toxicity of both P. acaciae and Wingea toxins was shown to be independent of RNA polymerase II Elongator, which is indispensable for zymocin action. Thus, P. acaciae and Wingea toxins differ in their mode of action from the G1-arresting zymocin. Fluorescence-activated cell sorting analysis and determination of budding indices have proved that such novel toxins mediate cell cycle arrest post-G1 during the S phase. Concomitantly, the DNA damage checkpoint kinase Rad53 is phosphorylated. As a mutant carrying the checkpoint-deficient allele rad53-11 displays toxin hypersensitivity, damage checkpoint activation apparently contributes to coping with toxin stress, rather than being functionally implemented in toxin action.     

WRN is required for ATM activation and the S-phase checkpoint in response to interstrand cross-link-induced DNA double-strand breaks

Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link–induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, γH2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link–induced collapsed replication forks.     

Two S-phase checkpoint systems, one involving the function of both BIME and Tyr15 phosphorylation of p34cdc2, inhibit NIMA and prevent premature mitosis.

We demonstrate that there are at least two S-phase checkpoint mechanisms controlling mitosis in Aspergillus. The first responds to the rate of DNA replication and inhibits mitosis via tyrosine phosphorylation of p34cdc2. Cells unable to tyrosine phosphorylate p34cdc2 are therefore viable but are unable to tolerate low levels of hydroxyurea and prematurely enter lethal mitosis when S-phase is slowed. However, if the NIMA mitosis-promoting kinase is inactivated then non-tyrosine-phosphorylated p34cdc2 cannot promote cells prematurely into mitosis. Lack of tyrosine-phosphorylated p34cdc2 also cannot promote mitosis, or lethality, if DNA replication is arrested, demonstrating the presence of a second S-phase checkpoint mechanism over mitotic initiation which we show involves the function of BIME. In order to overcome the S-phase arrest checkpoint over mitosis it is necessary both to prevent tyrosine phosphorylation of p34cdc2 and also to inactivate BIME. Lack of tyrosine phosphorylation of p34cdc2 allows precocious expression of NIMA during S-phase arrest, and lack of BIME then allows activation of this prematurely expressed NIMA by phosphorylation. The mitosis-promoting NIMA kinase is thus a target for S-phase checkpoint controls.