SEARCH · PubMed Health

Results for “Cell Cycle Checkpoints”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources

PubMed1996

Chemical induction of mitotic checkpoint override in mammalian cells results in aneuploidy following a transient tetraploid state.

Populations of tetraploid cells are found in a variety of human tumours where they may act as precursors of aneuploidy and tumorigenesis. Here we demonstrate the drug induction of tetraploid cells at mitosis by interference with cell cycle checkpoints and the coordination of mitotic events. Tetraploid cells result from mitotic exit in the absence of either chromosome segregation or cytokinesis. One class of agents that induces tetraploidy causes override of cell cycle checkpoints that require metaphase chromosome alignment as a pre-condition for initiating exit from mitosis. As a result cells exposed to such drugs progress partially through mitosis, but exit without chromosome segregation or cytokinesis. Inhibitors of microtubule assembly comprise a second class of agents that induce tetraploidy. Many cell types are incapable of maintaining indefinite mitotic arrest in the presence of microtubule inhibitors and finally exit mitosis without microtubule dependent chromosome segregation. Inhibitors of topoisomerase II represent a third class of drugs that induce tetraploidy at mitosis. By inhibiting DNA decatenation required for sister chromatid separation at the onset of anaphase such drugs block chromosome segregation. When topoisomerase II activity is inhibited, cells nonetheless reform nuclei and exit from mitosis without chromosome segregation. Finally, inhibition of cleavage furrow formation by agents such as cytochalasins represents a fourth mechanism of tetraploidization at mitosis. We find that when Chinese Hamster Ovary cells become tetraploid, regardless of which mechanism induces this state, they are genetically unstable and become aneuploid at the subsequent mitosis. In conclusion, the failure of mitotic checkpoint function can generate gross aneuploidy from which cells with an advantage for tumor growth may be selected.

2-Aminopurine↗

PubMed2003

Characterisation of telomerase immortalised normal human oesophageal squamous cells.

BACKGROUND AND AIMS: Oesophageal cell lines derived from malignancies have numerous genetic abnormalities and therefore are of limited value for studying the early events in carcinogenesis. Reported attempts to establish normal human oesophageal cell lines either have failed to achieve immortalisation or have achieved it by disrupting important cell functions. We have used telomerase technology to establish normal human oesophageal cell lines. METHODS: Endoscopic biopsy specimens of normal oesophageal squamous epithelium were trypsinised, dispersed into single cell suspensions, and cocultivated with ATCC Swiss 3T3 cells. Oesophageal cells were infected with the catalytic subunit of human telomerase (hTERT) using a defective retroviral vector. The integrity of cell cycle checkpoints was tested by measuring p53 response to UV irradiation, and p16 response to infection with H-RasGV12. Expression of a differentiation marker was tested by measuring involucrin response to calcium exposure. RESULTS: Cultures of uninfected oesophageal cells had weak telomerase activity at baseline but exhibited loss of telomerase activity and progressive telomere shortening before undergoing senescence between population doublings (PD) 40-45. In contrast, hTERT infected cells exhibited sustained telomerase activity and stabilisation of telomere length. These cells have reached PD 100 with no diminution in growth rate, while cell cycle checkpoint integrity and involucrin response to calcium exposure have remained intact. CONCLUSIONS: By introducing telomerase into normal human oesophageal squamous cells cocultivated with feeder layers, we have established a cell line that retains normal cell cycle checkpoints and normal differentiation markers. This cell line may be useful for studying the early events in oesophageal carcinogenesis.

3T3 Cells↗

PubMed1998

Mathematical model of the fission yeast cell cycle with checkpoint controls at the G1/S, G2/M and metaphase/anaphase transitions.

All events of the fission yeast cell cycle can be orchestrated by fluctuations of a single cyclin-dependent protein kinase, the Cdc13/Cdc2 heterodimer. The G1/S transition is controlled by interactions of Cdc13/Cdc2 and its stoichiometric inhibitor, Rum1. The G2/M transition is regulated by a kinase-phosphatase pair, Wee1 and Cdc25, which determine the phosphorylation state of the Tyr-15 residue of Cdc2. The meta/anaphase transition is controlled by interactions between Cdc13/Cdc2 and the anaphase promoting complex, which labels Cdc13 subunits for proteolysis. We construct a mathematical model of fission yeast growth and division that encompasses all three crucial checkpoint controls. By numerical simulations we show that the model is consistent with a broad selection of cell cycle mutants, and we predict the phenotypes of several multiple-mutant strains that have not yet been constructed.

Anaphase↗

PubMed2002

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. Through a combination of mutagenesis and phospho-specific antibodies, we have shown that serine at position 345 (S345) is phosphorylated in vivo in response to DNA damage, and that S345 phosphorylation is required for an intact checkpoint response. We have developed a kinase assay for Chk1, and have shown that basal Chk1 kinase activity is increased in response to DNA damage and that this increase, but not the basal activity, is dependent on S345. Furthermore, we show that S345 phosphorylation is required for Chk1 to associate with Rad24, a 14-3-3 protein, upon DNA damage. These results are consistent with a model whereby Chk1 phosphorylation results in increased Chk1 kinase activity that is necessary for both checkpoint delay and cellular survival following damage to the genome. These data are similar to observations made in mammalian cells and Xenopus oocyte extracts, suggesting that mechanisms leading to Chk1 activation have been conserved in evolution.

Amino Acid Sequence↗

PubMed2001

Checking out the G(2)/M transition.

Tight regulation of cell cycle progression is essential for the maintenance of genomic integrity. The orderly progression from one cell cycle phase to the other is mediated by timed activation of distinct cyclin/cdk complexes. For example, onset of mitosis is regulated by the activation of cyclin B/cdc2 and this event is controlled by several cell cycle checkpoints. Such checkpoints ensure that chromosome segregation does not occur in the case of unreplicated or damaged DNA, or misaligned chromosomes. Recently, new insights into the targets of the DNA damage checkpoint help to unravel more of the complex mechanisms of cell cycle checkpoints. This review focuses on the factors controlling the transition from G(2) phase to mitosis. Also, the pathways contributing to the DNA damage checkpoints in these phases of the cell cycle will be discussed.

Animals↗

PubMed1994

Role of retinoblastoma gene product in p53-mediated DNA damage response.

The cellular response to DNA-damaging agents involves the activation of cell cycle checkpoints. Checkpoints provide a transient delay in cell cycle progression, presumably to allow time for the cell to repair the damage. A most important checkpoint, active in the G1 phase of the cell cycle, is mediated by the p53 tumor suppressor gene product. To investigate the role of downstream components of the cell cycle machinery in p53-mediated G1 arrest, the possible involvement of the RB gene product was examined. Rb and p53 proteins were studied by immunoprecipitation and Western blotting experiments in the presence and absence of DNA-damaging treatment. The phosphorylation status of Rb was altered following DNA damage in p53 wild-type cell lines, but was not altered in p53 mutant cell lines, nor in cell lines where p53 function was abrogated by viral gene products. These findings indicate that Rb probably plays a role in the activation of the p53-mediated checkpoint.

Cell Cycle↗

PubMed1996

Genes involved in cell cycle G1 checkpoint control are frequently mutated in human melanoma metastases.

A common characteristic of cancer cells is unrestrained cell division. This may be caused by mutational changes in genes coding for components of cell cycle-controlling networks. Alterations in genes involved in G1 checkpoint control have been registered in many human tumours, and investigations from several laboratories show that such alterations, taken together, are the most frequent changes detected in cancer cells. The present paper describes mutational analysis by polymerase chain reaction-single-strand conformation polymorphism (PCR/SSCP) and nucleotide sequence analysis of the genes coding for the p15, p53 and N-ras proteins in 26 metastases from 25 melanoma patients. The registered mutation frequencies add together with previously registered mutations in p16 in the same patient samples to a substantial total frequency of 44% of patients with mutation in at least one of the investigated genes. These results show the occurrence of heterogeneous defects among components of the cell cycle controlling machinery in a human melanoma tumour sample collection and demonstrate that the total frequency of detected alterations increases with the number of cell cycle controlling genes included in the screening panel.

Carrier Proteins↗

PubMed2003

Regulation of meiotic progression by the meiosis-specific checkpoint kinase Mek1 in fission yeast.

During the eukaryotic cell cycle, accurate transmission of genetic information to progeny is ensured by the operation of cell cycle checkpoints. Checkpoints are regulatory mechanisms that block cell cycle progression when key cellular processes are defective or chromosomes are damaged. During meiosis, genetic recombination between homologous chromosomes is essential for proper chromosome segregation at the first meiotic division. In response to incomplete recombination, the pachytene checkpoint (also known as the meiotic recombination checkpoint) arrests or delays meiotic cell cycle progression, thus preventing the formation of defective gametes. Here, we describe a role for a meiosis-specific kinase, Mek1, in the meiotic recombination checkpoint in fission yeast. Mek1 belongs to the Cds1/Rad53/Chk2 family of kinases containing forkhead-associated domains, which participate in a number of checkpoint responses from yeast to mammals. We show that defects in meiotic recombination generated by the lack of the fission yeast Meu13 protein lead to a delay in entry into meiosis I owing to inhibitory phosphorylation of the cyclin-dependent kinase Cdc2 on tyrosine 15. Mutation of mek1(+) alleviates this checkpoint-induced delay, resulting in the formation of largely inviable meiotic products. Experiments involving ectopic overexpression of the mek1(+) gene indicate that Mek1 inhibits the Cdc25 phosphatase, which is responsible for dephosphorylation of Cdc2 on tyrosine 15. Furthermore, the meiotic recombination checkpoint is impaired in a cdc25 phosphorylation site mutant. Thus, we provide the first evidence of a connection between an effector kinase of the meiotic recombination checkpoint and a crucial cell cycle regulator and present a model for the operation of this meiotic checkpoint in fission yeast.

Amino Acid Sequence↗

PubMed2000

Hypersensitivity to radiation-induced non-apoptotic and apoptotic death in cell lines from patients with the ICF chromosome instability syndrome.

Immunodeficiency, centromeric region instability, and facial anomalies (ICF), a rare recessive chromosome instability syndrome, involves the loss of DNA methyltransferase 3B activity and the consequent hypomethylation of a small portion of the genome. We demonstrate for the first time that ICF cells are strongly hypersensitive to a genotoxic agent, namely, ionizing radiation. However, unlike cell lines from patients with ataxia telangiectasia or Nijmegen breakage syndrome, chromosome instability syndromes also associated with unusual sensitivity to ionizing radiation, ICF cells did not show any deficiencies in their cell cycle checkpoints. ICF lymphoblastoid cell lines demonstrated increased apoptosis, long-term cell cycle arrest, and loss of viability in clonogenicity assays after irradiation compared to analogous normal cell lines. Also, the ICF cell lines were subject to high frequencies of rapid non-apoptotic cell death upon irradiation but not to abnormally high levels of radiation-induced, cytogenetically detectable chromosome abnormalities. ICF-associated undermethylation of some regulatory gene(s) might lead to an exaggerated response to radiation-induced breaks in DNA yielding increased rates of cell death and irreversible cell cycle arrest. As a defense against their frequent spontaneous breaks in chromosomes 1 and 16, ICF patients may be abnormally prone to chromosome break-induced apoptosis, non-apoptotic cell death, and permanent cell cycle arrest so as to minimize the number of cycling cells with spontaneous rearrangements. A similarly increased cell death and cycle-arrest response to chromosome breaks due to cancer-linked DNA hypomethylation might occur during carcinogenesis.

Apoptosis↗

PubMed1996

Requirement for Ku80 in growth and immunoglobulin V(D)J recombination.

The DNA-dependent protein kinase (DNA-PK) is a mammalian serine/threonine kinase that is implicated in the repair of DNA double-strand breaks, DNA replication, transcription, and V(D)J recombination. To determine the role of the DNA-binding subunit of DNA-PK in vivo, we targeted Ku80 in mice. In mutant mice, T and B lymphocyte development is arrested at early progenitor stages and there is a profound deficiency in V(D)J rearrangement. Although Ku80-/- mice are viable and reproduce, they are 40-60% of the size of littermate controls. Consistent with this growth defect, fibroblasts derived from Ku80-/- embryos showed an early loss of proliferating cells, a prolonged doubling time, and intact cell-cycle checkpoints that prevented cells with damaged DNA from entering the cell-cycle. The unexpected growth phenotype suggests a new and important link between Ku80 and growth control.

Animals↗

PubMed1998

Chromosomal instability is correlated with telomere erosion and inactivation of G2 checkpoint function in human fibroblasts expressing human papillomavirus type 16 E6 oncoprotein.

Cell cycle checkpoints and tumor suppressor gene functions appear to be required for the maintenance of a stable genome in proliferating cells. In this study chromosomal destabilization was monitored in relation to telomere structure, lifespan control and G2 checkpoint function. Replicative senescence was inactivated in secondary cultures of human skin fibroblasts by expressing the human papillomavirus type 16 (HPV-16) E6 oncoprotein to inactivate p53. Chromosome aberrations were enumerated during in vitro aging of isogenic control (F5neo) and HPV-16E6-expressing (F5E6) fibroblasts. We found that structural and numerical aberrations in chromosomes were significantly increased in F5E6 cells during aging in vitro and fluorescence in situ hybridization (FISH) analysis using chromosome-specific probes demonstrated the occurrence of rearrangements involving chromosome 4 and 6 in genetically unstable F5E6 cells. Flow cytometry and karyotypic analyses revealed increased polyploidy and aneuploidy in F5E6 cells only at passages > 16, although these cells displayed defective mitotic spindle checkpoint function associated with inactivation of p53 at passages 5 and 16. G2 checkpoint function was confirmed to be gradually but progressively inactivated during in vitro aging of E6-expressing cells. Aging of F5neo fibroblasts was documented during in vitro passaging by induction of a senescence-associated marker, pH 6.0 lysosomal beta-galactosidase. F5E6 cells displayed extension of in vitro lifespan and did not induce beta-galactosidase at high passage. Erosion of telomeres during in vitro aging of telomerase-negative F5neo cells was demonstrated by Southern hybridization and by quantitative FISH analysis on an individual cell level. Telomeric signals diminished continuously as F5neo cells aged in vitro being reduced by 80% near the time of replicative senescence. Telomeric signals detected by FISH also decreased continuously during aging of telomerase-negative F5E6 cells, but telomeres appeared to be stabilized at passage 34 when telomerase was expressed. Chromosomal instability in E6-expressing cells was correlated (P < 0.05) with both loss of telomeric signals and inactivation of G2 checkpoint function. The results suggest that chromosomal stability depends upon a complex interaction among the systems of telomere length maintenance and cell cycle checkpoints.

Cell Line↗

PubMed2000

Regulation of cell death by the Abl tyrosine kinase.

The c-abl proto-oncogene encodes a protein tyrosine kinase that is distributed in the nucleus and the cytoplasm of proliferating cells. In the nucleus, c-Abl activity is negatively regulated by the retinoblastoma protein (RB) and positively regulated by DNA damage signals. Activation of the c-Abl kinase by DNA damage requires the function of ATM, which regulates cell cycle checkpoint, DNA repair and apoptosis in response to DNA damage. Cells lacking c-Abl can activate cell cycle checkpoints and DNA repair, but show defects in apoptosis. The apoptosis defect of c-Abl deficient cells is correlated with a defect in the induction and activation of p73, which is a functional homologue of the p53 tumor suppressor protein and has pro-apoptotic activity. The inhibition of c-Abl by RB is consistent with RB's ability to block apoptosis; while the activation of c-Abl by ATM is consistent with ATM's ability to activate cell death. The oncogenic Bcr-Abl tyrosine kinase is a potent inhibitor of apoptosis, and it is retained exclusively in the cytoplasm of transformed cells. Interestingly, when Bcr-Abl is trapped inside of the nucleus through a combined disruption of its cytoplasmic retention and its nuclear export, this oncogenic Abl kinase induces apoptosis. Taken together, the current results support a role for the nuclear c-Abl tyrosine kinase in the regulation of apoptosis. Whether the cytoplasmic c-Abl kinase can actively inhibit apoptosis remains to be determined; however, a deliberate retention of c-Abl in the cytoplasm could potentially contribute to the attenuation of apoptosis response.

Apoptosis↗

PubMed2003

Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents.

UV and ionizing radiation (IR) activate DNA damage checkpoints and induce Cdc25A degradation (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429; Falck, J., Mailand, N., Syljuasen, R. G., Bartek, J., and Lukas J. (2001) Nature 410, 842-847). The degradation of Cdc25A is abrogated by caffeine, which implicates Chk1 as the potential mediator (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429). However, the involvement of Chk1 is far from clear, because caffeine is a rather nonspecific inhibitor of the ATR/Chk1 signaling pathway. Additionally, it is not known whether DNA-damaging drugs commonly used in chemotherapy, which may activate different signal transduction pathways than UV or IR, also confer Cdc25A degradation. Herein, we show that camptothecin and doxorubicin, two widely used topoisomerase inhibitors conferring S and G2 arrest, respectively, cause the degradation of Cdc25A. Using a small interfering RNA that enables the specific elimination of Chk1 expression, we show that the observed proteolysis of Cdc25A is mediated through Chk1. Moreover, Cdc25A overexpression abrogates the Chk1-mediated degradation and overcomes the doxorubicin-induced G2 arrest through dephosphorylation and activation of Cdc2/Cdk1 in a dose-dependent manner. These results suggest that: (a) Cdc25A is involved in the G2/M transition in addition to its commonly accepted effect on G1/S progression, and (b) Chk1 mediates both S and G2 checkpoint and is thus a more ubiquitous cell cycle checkpoint mediator than previously thought.

Antineoplastic Agents, Phytogenic↗

PubMed1998

Attenuation of G1 checkpoint function by the non-genotoxic carcinogen phenobarbital.

Non-genotoxic chemical carcinogens are capable of inducing tumors in rodents without interacting with or directly altering the genetic material. Since a preponderance of evidence suggests that cancer results from the accumulation of genetic alterations, the mechanisms by which many non-genotoxic carcinogens induce genotoxic events remain unclear. The present study investigated whether the mitogenic, non-genotoxic carcinogen phenobarbital (PB) could alter cell-cycle checkpoint controls, thereby indirectly leading to the accumulation of genetic damage. Initial studies involved characterizing cell-cycle checkpoint responses to DNA damage in freshly isolated B6C3F1 mouse hepatocytes. These cells responded to bleomycin-induced DNA damage by arresting in G1 and G2. Cell-cycle arrest was coupled with p53 protein induction; however, p21WAF1 protein levels remained unchanged. Studies that utilized hepatocytes isolated from C57BL p53-/- mice showed that the DNA damage-induced G1 cell-cycle arrest was dependent on p53 function, but cell-cycle arrest in G2 was not affected by loss of p53. PB was able to delay and attenuate the G1 checkpoint response without altering G2 checkpoint function. A reduction in p53 protein, but not transcript levels, was observed in hepatocytes exposed to PB. Additionally, PB delayed and attenuated p53 protein induction during DNA damage, which suggests that changes in the p53 protein may be contributing to the attenuated G1 checkpoint response caused by PB. Altered G1 checkpoint function represents an epigenetic mechanism by which phenobarbital may prevent the detection and repair of DNA damage and indirectly increase the frequency of genotoxic events above that occurring spontaneously. Abrogation of checkpoint controls may, thus, play an important mechanistic role in mitogenic, non-genotoxic chemical carcinogenesis.

Animals↗

PubMed2001

ATM-dependent phosphorylation of human Rad9 is required for ionizing radiation-induced checkpoint activation.

ATM (ataxia-telangiectasia-mutated) is a Ser/Thr kinase involved in cell cycle checkpoints and DNA repair. Human Rad9 (hRad9) is the homologue of Schizosaccharomyces pombe Rad9 protein that plays a critical role in cell cycle checkpoint control. To examine the potential signaling pathway linking ATM and hRad9, we investigated the modification of hRad9 in response to DNA damage. Here we show that hRad9 protein is constitutively phosphorylated in undamaged cells and undergoes hyperphosphorylation upon treatment with ionizing radiation (IR), ultraviolet light (UV), and hydroxyurea (HU). Interestingly, hyperphosphorylation of hRad9 induced by IR is dependent on ATM. Ser(272) of hRad9 is phosphorylated directly by ATM in vitro. Furthermore, hRad9 is phosphorylated on Ser(272) in response to IR in vivo, and this modification is delayed in ATM-deficient cells. Expression of hRad9 S272A mutant protein in human lung fibroblast VA13 cells disturbs IR-induced G(1)/S checkpoint activation and increased cellular sensitivity to IR. Together, our results suggest that the ATM-mediated phosphorylation of hRad9 is required for IR-induced checkpoint activation.

Ataxia Telangiectasia↗

PubMed2000

Involvement of apoptosis in mitomycin C hypersensitivity of Chinese hamster cell mutants.

To elucidate the mechanisms of the mammalian cell defense against cross-linking agents, we studied previously cellular responses to mitomycin C (MMC) treatment in two MMC-hypersensitive hamster cell mutants' V-H4 and V-C8, as well as their parental cell line V79. In the present report, we investigated whether alterations in cell cycle checkpoints and induction of apoptosis could be responsible for the MMC hypersensitivity of the V-H4 and V-C8 mutant cell lines. First, we found that parental and mutant cells exhibited similar cell cycle responses to MMC concentrations of equivalent cytotoxicity, arguing against a defective cell cycle checkpoint in hypersensitive cell lines. In contrast, we showed that mutant cells underwent greater levels of apoptosis following MMC treatment than parental cells. These findings indicate that increased induction of apoptosis contributes to the hypersensitivity of V-H4 and V-C8 cells to the growth inhibitory effect of MMC. This differential apoptotic response was observed with both equimolar and equitoxic MMC doses and was specific to the cross-linking agent MMC, suggesting that control of the apoptotic process is altered in both MMC-hypersensitive mutants. The defective genes in V-H4 and V-C8 cells would then function in the regulation of an apoptotic pathway triggered by MMC-induced damage and independent of p53-mediated transcription.

Animals↗

PubMed1996

Spontaneous p53 mutation in murine mesothelial cells: increased sensitivity to DNA damage induced by asbestos and ionizing radiation.

The p53 gene regulates the G1 cell cycle checkpoint in response to DNA damage. A primary murine mesothelial cell line (D9) spontaneously acquired a point mutation at codon 135 in exon 5 of the p53 gene, resulting in substitution of alanine for proline; early passage D9 cells expressed wild-type p53. The growth rate of late passage D9 cells that acquired the p53 mutation was increased compared to that of early passage cells; however, this mutation was not sufficient to confer tumorigenicity to this cell line. Mammalian cells that express wild-type p53 show a transient arrest in G1 after exposure to ionizing radiation. Early passage D9 cells showed a G1 arrest following ionizing radiation, while late passage D9 cells arrested in G2 or mitosis. The clastogenic effects of ionizing radiation can be demonstrated by the cytokinesis-arrested micronucleus assay. Following treatment with cytochalasin B to arrest cytokinesis, ionizing radiation induced micronuclei in 50% of late passage D9 cells compared to 15% of early passage cells. After exposure to 15 micrograms/cm2 of crocidolite asbestos fibers, 18% of late passage cells had micronuclei compared to 4% of early passage cells. It is hypothesized that loss of the G1 cell cycle checkpoint contributes to genetic instability in murine mesothelial cells.

Animals↗

PubMed2001

Role of fission yeast primase catalytic subunit in the replication checkpoint.

To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1(+) and spp2(+) (S. pombe primase 1 and 2). spp1(+) encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polalpha to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1(+) gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1(+) exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1(+) reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polalpha-primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polalpha has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polalpha. Together, these have led us to propose that suboptimal cellular levels of polalpha-primase complex due to the allele-specific mutations of Spp1 might not allow Polalpha to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.

Amino Acid Sequence↗