Budding Yeast DNA Damage Adaptation Mutants Exhibit Defects in Mitotic Exit

In the presence of double strand breaks, DNA damage checkpoint halts cell cycle progression. However, cells ultimately escape the checkpoint arrest and re-enter cell cycle in the presence of irreparable DNA damage. cdc5-ad was identified as a mutant that fails to adapt to the cell cycle arrest induced by DNA damage checkpoint. In budding yeast, Cdc5 protein kinase is a component of both MEN and FEAR pathways that are required for mitotic exit. It remains unclear whether the adaptation defect of cdc5-ad mutant cells is related to the function of Cdc5 in mitotic exit. Here we present evidence indicating that cdc5-ad mutant cells exhibit defects in mitotic exit. cdc5-ad mutant cells are sensitive to high dosage of Amn1, a negative regulator of MEN. It also shows synthetic growth defects with mutants in MEN pathway. Moreover, mutants in FEAR pathway exhibit defects in DNA damage adaptation. Thus, we conclude that the compromised mitotic exit pathway contributes to DNA damage adaptation defects in cdc5-ad mutant cells.     

Identification of Residues in Fission Yeast and Human P34(cdc2) Required for S-M Checkpoint Control

In fission yeast, regulation of p34(cdc2) plays an important role in the checkpoint coupling mitosis to completion of DNA replication. The cdc2 mutations cdc2-3w (C67Y) and cdc2-4w (C67F) abolish checkpoint control without seriously affecting normal cell proliferation. However the molecular basis of this phenotype is not known. To better understand the role of p34(cdc2) in checkpoint control, we have screened for more mutations in Schizosaccharomyces pombe cdc2 with this phenotype. We have isolated cdc2-3w and cdc2-4w, as well as three new cdc2 alleles: cdc2-6w (N66I), cdc2-7w (E8V) and cdc2-8w (K9E). The altered residues map to two different regions on opposite faces of the protein, suggesting that the interaction between p34(cdc2) and components of the checkpoint pathway may be complex. In contrast to cdc2-3w and cdc2-4w, the new mutations alter residues that are conserved between the fission yeast cdc2(+) and other cdks, including the human CDC2 protein. Expression of the equivalent human CDC2 mutants in fission yeast abolishes checkpoint control, suggesting that these residues could be involved in checkpoint-dependent regulation of other eukaryotic cdks.     

The bacterial replication initiator DnaA. DnaA and oriC,the bacterial mode to initiate DNA replication

The initiation of replication is the central event in the bacterial cell cycle. Cells control the rate of DNA synthesis by modulating the frequency with which new chains are initiated, like all macromolecular synthesis. The end of the replication cycle provides a checkpoint that must be executed for cell division to occur. This review summarizes recent insight into the biochemistry, genetics and control of the initiation of replication in bacteria, and the central role of the initiator protein DnaA.     

Cell Cycle Arrest of Cdc Mutants and Specificity of the Rad9 Checkpoint

In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G(2) phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G(2) phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G(2) phase.     

Segregation of unreplicated chromosomes in Saccharomyces cerevisiae reveals a novel G1/M-phase checkpoint.

Saccharomyces cerevisiae dbf4 and cdc7 cell cycle mutants block initiation of DNA synthesis (i.e., are iDS mutants) at 37 degrees C and arrest the cell cycle with a 1C DNA content. Surprisingly, certain dbf4 and cdc7 strains divide their chromatin at 37 degrees C. We found that the activation of the Cdc28 mitotic protein kinase and the Dbf2 kinase occurred with the correct relative timing with respect to each other and the observed division of the unreplicated chromatin. Furthermore, the division of unreplicated chromatin depended on a functional spindle. Therefore, the observed nuclear division resembled a normal mitosis, suggesting that S. cerevisiae commits to M phase in late G1 independently of S phase. Genetic analysis of dbf4 and cdc7 strains showed that the ability to restrain mitosis during a late G1 block depended on the genetic background of the strain concerned, since the dbf4 and cdc7 alleles examined showed the expected mitotic restraint in other backgrounds. This restraint was genetically dominant to lack of restraint, indicating that an active arrest mechanism, or checkpoint, was involved. However, none of the previously described mitotic checkpoint pathways were defective in the iDS strains that carry out mitosis without replicated DNA, therefore indicating that the checkpoint pathway that arrests mitosis in iDS mutants is novel. Thus, spontaneous strain differences have revealed that S. cerevisiae commits itself to mitosis in late G1 independently of entry into S phase and that a novel checkpoint mechanism can restrain mitosis if cells are blocked in late G1. We refer to this as the G1/M-phase checkpoint since it acts in G1 to restrain mitosis.     

Ectopic Expression of cdc2/cdc28 Kinase Subunit Homo sapiens 1 Uncouples Cyclin B Metabolism from the Mitotic Spindle Cell Cycle Checkpoint

Primary human fibroblasts arrest growth in response to the inhibition of mitosis by mitotic spindle-depolymerizing drugs. We show that the mechanism of mitotic arrest is transient and implicates a decrease in the expression of cdc2/cdc28 kinase subunit Homo sapiens 1 (CKsHs1) and a delay in the metabolism of cyclin B. Primary human fibroblasts infected with a retroviral vector that drives the expression of a mutant p53 protein failed to downregulate CKsHs1 expression, degraded cyclin B despite the absence of chromosomal segregation, and underwent DNA endoreduplication. In addition, ectopic expression of CKsHs1 interfered with the control of cyclin B metabolism by the mitotic spindle cell cycle checkpoint and resulted in a higher tendency to undergo DNA endoreduplication. These results demonstrate that an altered regulation of CKsHs1 and cyclin B in cells that carry mutant p53 undermines the mitotic spindle cell cycle checkpoint and facilitates the development of aneuploidy. These data may contribute to the understanding of the origin of heteroploidy in mutant p53 cells.     

Interaction between cdc13+ and cdc2+ in the control of mitosis in fission yeast; dissociation of the G1 and G2 roles of the cdc2+ protein kinase

A cold-sensitive (cs) allele of cdc2, a gene that acts in both the G1 and G2 phases of the fission yeast cell cycle, has been isolated by classical mutagenesis. Further mutagenesis of a cdc2cs strain yielded an extragenic suppressor that rescued the cs cell cycle defect but simultaneously conferred a temperature-sensitive (ts) cdc phenotype. This suppressor mutation was shown to be an allele of cdc13, a previously identified gene. A variety of allele-specific interactions between cdc2 and cdc13 were discovered. These included suppression of cdc13ts alleles by introduction of the cdc2+ gene on a multi-copy plasmid vector. cdc13+ is required in G2 for mitotic initiation and was shown to play no role in the G1 phase of the cell cycle. cdc2+, however, is essential in G1 for DNA replication and in G2 for mitosis. The newly isolated cs allele of cdc2 that is rescued by a ts allele of cdc13 is defective only in its G2 function. cdc13+ cooperates with cdc2+ in the initiation of mitosis but not in the regulation of DNA replication. We propose that the cdc13+ gene product might be a G2-specific substrate of the cdc2+ protein kinase.     

Regulation of the initiation step of DNA replication by cyclin-dependent kinases

Cyclin-dependent kinases (CDKs) play a central role in the regulation of cell cycle progression in eukaryotes. The onset of S phase, the initiation of chromosomal DNA replication, is a major cell cycle event that is regulated by CDKs. Eukaryotic chromosomal DNA replication is highly regulated and occurs as a two-step reaction. The first reaction, known as licensing, is essential for DNA replication by making cell replication competent and occurs in G1 phase. Once cells enter S phase, licensed chromosomes initiate DNA replication through the action of two conserved protein kinases, S phase-specific CDK and Cdc7-Dbf4 (or Dbf4-dependent kinase). Our understanding of the regulatory mechanisms of DNA replication in model eukaryotes has advanced considerably in the past decade. In this review, we overview the regulation of DNA replication in the eukaryotic cell cycle, focusing specifically on how CDKs regulate the initiation step of DNA replication.     

Polo-like kinases and Chk2 at the interface of DNA damage checkpoint pathways and mitotic regulation

The cell cycle controls processes of DNA replication and segregation of replicated DNA into two daughter cells. These processes are coordinated by multiple signaling pathways, which employ many protein kinases. The members of the family of Polo-like protein kinases are among these key cell cycle regulators. In response to DNA damage and inhibited DNA replication, DNA structure checkpoints delay cell cycle progression to provide cells with time for repair of damaged DNA and protect it from more severe damage. These effects are achieved by affecting key players of the basic cell cycle regulation of the cells with damaged DNA. This review is focused on the interplay between Chk2, a bona fide checkpoint protein kinase, and Polo-like kinases.     

Reovirus-Induced ς1s-Dependent G2/M Phase Cell Cycle Arrest Is Associated with Inhibition of p34cdc2

Serotype 3 reoviruses inhibit cellular proliferation by inducing a G(2)/M phase cell cycle arrest. Reovirus-induced G(2)/M phase arrest requires the viral S1 gene-encoded sigma1s nonstructural protein. The G(2)-to-M transition represents a cell cycle checkpoint that is regulated by the kinase p34(cdc2). We now report that infection with serotype 3 reovirus strain Abney, but not serotype 1 reovirus strain Lang, is associated with inhibition and hyperphosphorylation of p34(cdc2). The sigma1s protein is necessary and sufficient for inhibitory phosphorylation of p34(cdc2), since a viral mutant lacking sigma1s fails to hyperphosphorylate p34(cdc2) and inducible expression of sigma1s is sufficient for p34(cdc2) hyperphosphorylation. These studies establish a mechanism by which reovirus can perturb cell cycle regulation.     

Claspin: Timing the Cell Cycle Arrest When the Genome is Damaged

DNA damage checkpoints maintain genomic integrity by delaying cell cycle progression in response to genotoxic stress and stalled replication forks. One central pathway in the checkpoint response is the ATR-Chk1 pathway, in which, upon DNA damage, ATR phosphorylates and activates the effector kinase Chk1. This process depends on the adaptor protein Claspin that bridges ATR and Chk1. Once the damage is repaired, this pathway must somehow be switched off to allow the cell to continue the cell division process, an event known as checkpoint recovery. Polo-like kinase 1 (Plk1) plays a central role during checkpoint recovery. Interestingly, the Xenopus homologue of Plk1, Plx1, is able to bind and phosphorylate Claspin, releasing it from DNA and thereby contributing to Chk1 inactivation. Moreover, it was recently demonstrated that Claspin levels are controlled by proteasomal degradation, and this is regulated by Plk1. Importantly, Plk1-mediated proteosomal degradation of Claspin appears to be essential for checkpoint recovery. Here we review these recent findings and discuss the mechanisms of checkpoint regulation by Claspin.     

Cdc2-kinases,cyclins, and the switch from proliferation to polyploidization

Summary Almost all organisms, from protists to humans, and from algae to orchids, display somatic polyploidy, including polyteny. In insects and higher plants, nearly all normal, differentiated cells are polyploid, corresponding to the majority of living matter. So far, no universal mechanism controlling the switch from proliferation to polyploidization has been proposed. However, recent progress in understanding regulation of the mitotic cell cycle by protein kinases and cyclins allows some unifying ideas which can be experimentally tested to be put forward. The key events are the abolishment of the dependence of DNA replication on mitosis, and changes in the expression and activity of the complexes formed by cyclin-dependent kinases and cyclins. In addition, repression of further cell cycle control genes may allow underreplication of DNA, characteristic of endo-cycles in many insects and angiosperms. Change to a different checkpoint may be responsible for gene amplification. The switch in cell cycle control is developmentally regulated by signal transduction cascades, which are briefly discussed. Polyploidy is also known from many cancers, where genetic and metabolic disturbances lead to a similar switch to that in normal cells. The related literature is reviewed and some possible lines of future research are suggested.Abbreviations CAK p34^cdc2-activating kinase - cdc2 cell division cycle gene inSchizosaccharomyces pombe (fission yeast), named cdk1 in mammals - CDKs cyclin-dependent kinases - cdk2 S-phase specific CDK gene in higher organisms - MAP kinase mitogen-activated protein kinase - MAPs microtubule-associated proteins - MPF maturation (or mitosis) promoting factor - p34^cdc2 mitosis specific protein kinase     

A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.     

Cmk2, a novel serine/threonine kinase in fission yeast

The cmk2 gene of Schizosaccharomyces pombe encodes a 504 amino acid protein kinase with sequence homology with the calmodulin-dependent protein kinase family. The cmk2(+) gene is not essential for cell viability but overexpression of cmk2(+) blocks the cell cycle at G2 phase and this inhibition is cdc2-dependent. The Cmk2 is a cytoplasmic protein expressed in a cell cycle-dependent manner, peaking at the G1/S boundary. Overexpression of Cmk2 suppresses fission yeast DNA replication checkpoint defects but not DNA damage checkpoint defects, suggesting that the G2 cell cycle arrest mediated by high levels of Cmk2 provides sufficient time to correct DNA replication alterations.     

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.     

RAD53 regulates DBF4 independently of checkpoint function in Saccharomyces cerevisiae

The Cdc7p and Dbf4p proteins form an active kinase complex in Saccharomyces cerevisiae that is essential for the initiation of DNA replication. A genetic screen for mutations that are lethal in combination with cdc7-1 led to the isolation of seven lsd (lethal with seven defect) complementation groups. The lsd7 complementation group contained two temperature-sensitive dbf4 alleles. The lsd1 complementation group contained a new allele of RAD53, which was designated rad53-31. RAD53 encodes an essential protein kinase that is required for the activation of DNA damage and DNA replication checkpoint pathways, and that is implicated as a positive regulator of S phase. Unlike other RAD53 alleles, we demonstrate that the rad53-31 allele retains an intact checkpoint function. Thus, the checkpoint function and the DNA replication function of RAD53 can be functionally separated. The activation of DNA replication through RAD53 most likely occurs through DBF4. Two-hybrid analysis indicates that the Rad53p protein binds to Dbf4p. Furthermore, the steady-state level of DBF4 message and Dbf4p protein is reduced in several rad53 mutant strains, indicating that RAD53 positively regulates DBF4. These results suggest that two different functions of the cell cycle, initiation of DNA replication and the checkpoint function, can be coordinately regulated through the common intermediate RAD53.     

Cyclin A- and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts.

Cyclins are proteins which are synthesized and degraded in a cell cycle-dependent fashion and form integral regulatory subunits of protein kinase complexes involved in the regulation of the cell cycle. The best known catalytic subunit of a cyclin-dependent protein kinase complex is p34cdc2. In the cell, cyclins A and B are synthesized at different stages of the cell cycle and induce protein kinase activation with different kinetics. The kinetics of activation can be reproduced and studied in extracts of Xenopus eggs to which bacterially produced cyclins are added. In this paper we report that in egg extracts, both cyclin A and cyclin B associate with and activate the same catalytic subunit, p34cdc2. In addition, cyclin A binds a less abundant p33 protein kinase related to p34cdc2, the product of the cdk2/Eg1 gene. When complexed to cyclin B, p34cdc2 is subject to transient inhibition by tyrosine phosphorylation, producing a lag between the addition of cyclin and kinase activation. In contrast, p34cdc2 is only weakly tyrosine phosphorylated when bound to cyclin A and activates rapidly. This finding shows that a given kinase catalytic subunit can be regulated in a different manner depending on the nature of the regulatory subunit to which it binds. Tyrosine phosphorylation of p34cdc2 when complexed to cyclin B provides an inhibitory check on the activation of the M phase inducing protein kinase, allowing the coupling of processes such as DNA replication to the onset of metaphase. Our results suggest that, at least in the early Xenopus embryo, cyclin A-dependent protein kinases may not be subject to this checkpoint and are regulated primarily at the level of cyclin translation.     

14-3-3 gamma is required to enforce both the incomplete S phase and G2 DNA damage checkpoints

Checkpoint pathways inhibit mitotic progression by inducing the phosphorylation of serine 216 in cdc25C resulting in the generation of a 14-3-3 binding site on cdc25C. Two 14-3-3 isoforms, 14-3-3ε and 14-3-3γ form a complex with cdc25C and inhibit cdc25C function. To examine the contribution of 14-3-3γ to checkpoint regulation, the expression of 14-3-3γ was inhibited in HCT116 cells using vector based RNA interference. A transient reduction in the expression of 14-3-3γ in HCT116 cells resulted in an override of both the incomplete S phase and the G2 DNA damage checkpoint. A 14-3-3γ knockdown clone also showed an override of both checkpoint pathways. These phenotypes were reversed upon expression of a shRNA resistant 14-3-3γ cDNA. Override of the G2 DNA damage checkpoint pathway was accompanied by a decrease in the levels of inhibitory phosphorylation on cdc25C and cdk1. However, there was no difference in the γ-H2AX foci formation and levels of phospho-chk1 and phospho-chk2, suggesting that activation of the DNA damage checkpoint response and subsequent activation of the checkpoint kinases Chk1 and Chk2 was not perturbed. These results suggest that the override of checkpoint observed in 14-3-3γ knockdown cells is due to failure to inhibit cdc25C function.