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PubMed2005

Differential impact of diverse anticancer chemotherapeutics on the Cdc25A-degradation checkpoint pathway.

When exposed to DNA-damaging insults such as ionizing radiation (IR) or ultraviolet light (UV), mammalian cells activate checkpoint pathways to halt cell cycle progression or induce cell death. Here we examined the ability of five commonly used anticancer drugs with different mechanisms of action to activate the Chk1/Chk2-Cdc25A-CDK2/cyclin E cell cycle checkpoint pathway, previously shown to be induced by IR or UV. Whereas exposure of human cells to topoisomerase inhibitors camptothecin, etoposide, or adriamycin resulted in rapid (within 1 h) activation of the pathway including degradation of the Cdc25A phosphatase and inhibition of cyclin E/CDK2 kinase activity, taxol failed to activate this checkpoint even after a prolonged treatment. Unexpectedly, although the alkylating agent cisplatin also induced degradation of Cdc25A (albeit delayed, after 8-12 h), cyclin E/CDK2 activity was elevated and DNA synthesis continued, a phenomena that correlated with increased E2F1 protein levels and consequently enhanced expression of cyclin E. These results reveal a differential impact of various classes of anticancer chemotherapeutics on the Cdc25A-degradation pathway, and indicate that the kinetics of checkpoint induction, and the relative balance of key components within the DNA damage response network may dictate whether the treated cells arrest their cell cycle progression.

Antibiotics, Antineoplastic↗
PubMed1997

DNA-replication checkpoint control at the Drosophila midblastula transition.

Embryogenesis is typically initiated by a series of rapid mitotic divisions that are under maternal genetic control. The switch to zygotic control of embryogenesis at the midblastula transition is accompanied by significant increases in cell-cycle length and gene transcription, and changes in embryo morphology. Here we show that mutations in the grapes (grp) checkpoint 1 kinase homologue in Drosophila block the morphological and biochemical changes that accompany the midblastula transition, lead to a continuation of the maternal cell-cycle programme, and disrupt DNA-replication checkpoint control of cell-cycle progression. The timing of the midblastula transition is controlled by the ratio of nuclei to cytoplasm (the nucleocytoplasmic ratio), suggesting that this developmental transition is triggered by titration of a maternal factor by the increasing mass of nuclear material that accumulates during the rapid embryonic mitoses. Our observations support a model for cell-cycle control at the midblastula transition in which titration of a maternal component of the DNA-replication machinery slows DNA synthesis and induces a checkpoint-dependent delay in cell-cycle progression. This delay may allow both completion of S phase and transcription of genes that initiate the switch to zygotic control of embryogenesis.

Animals↗
PubMed2005

Phosphorylation of Plk1 at S137 and T210 is inhibited in response to DNA damage.

Polo-like kinase 1 (Plk1) regulates multiple processes during mitosis. Plk1 is activated by phosphorylation at the G2/M phase boundary. Active Plk1 is involved in promotion of mitotic entry through activation of Cdc25C, and through nuclear import of cyclin B1 that together activate Cdc2/cyclin B kinase. In earlier work, phosphopeptide mapping identified several phosphorylation sites in Plk1. Mutational analysis pinpointed threonine 210, which is located in the activation loop of the kinase domain, as the major activation site of Plk1. In response to DNA damage, ATM/ATR-dependent checkpoint pathways inhibit Plk1 activity. Insensitivity of Plk1T210D, a constitutively active mutant, to DNA damage-induced inhibition of Plk1 indicates that regulation of Plk1 phosphorylation is a potential target of DNA damage checkpoints. In the present paper, we report that in vivo phosphorylation of Plk1 at serine 137 (S137) and threonine 210 (T210) occurs in mitosis. DNA damage prevents phosphorylation of Plk1 at both S137 and T210 in asynchronous cells but not in mitotic cells. Inhibitors of ATM/ATR and Chk1/Chk2 protein kinases avert the inhibition of Plk1 phosphorylation in response to DNA damage. These data suggest a participation of DNA damage checkpoints in regulation of the signaling pathways upstream of Plk1.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2004

CHK1 and CHK2 are differentially involved in mismatch repair-mediated 6-thioguanine-induced cell cycle checkpoint responses.

The DNA mismatch repair (MMR) system plays an important role in mediating a G2-M checkpoint arrest and subsequent cell death following treatment with a variety of chemotherapeutic agents. In this study, using 6-thioguanine (6-TG) as a mismatch-inducing drug, we examine the role of ataxia telangiectasia mutated (ATM)/CHK2 and ATM and Rad-3 related (ATR)/CHK1 signaling pathways in MMR-mediated cell cycle responses in MMR-proficient human colorectal cancer RKO cells. We show that, in response to 6-TG (3 micromol/L x 24 hours), activating phosphorylation of CHK1 at Ser317 [CHK1(pS317)] and CHK2 at Thr68 [CHK2(pT68)] are induced differentially during a prolonged course (up to 6 days) of MMR-mediated cell cycle arrests following 6-TG treatment, with CHK1(pS317) being induced within 1 day and CHK2(pT68) being induced later. Using chemical inhibitors and small interfering RNA of the signaling kinases, we show that a MMR-mediated 6-TG-induced G2 arrest is ATR/CHK1 dependent but ATM/CHK2 independent and that ATR/CHK1 signaling is responsible for both initiation and maintenance of the G2 arrest. However, CHK2(pT68) seems to be involved in a subsequent tetraploid G1 arrest, which blocks cells that escape from the G2-M checkpoint following 6-TG treatment. Furthermore, we show that CHK2 is hyperphosphorylated at later times following 6-TG treatment and the phosphorylation of CHK2 seems to be ATM independent but up-regulated when ATR or CHK1 is reduced. Thus, our data suggest that CHK1(pS317) is involved in a MMR-mediated 6-TG-induced G2 arrest, whereas CHK2(pT68) seems to be involved in a subsequent tetraploid G1-S checkpoint. The two signaling kinases seem to work cooperatively to ensure that 6-TG damaged cells arrest at these cell cycle checkpoints.

Antimetabolites, Antineoplastic↗
PubMed2005

Human Rad9 is required for the activation of S-phase checkpoint and the maintenance of chromosomal stability.

In response to DNA damage or replication block, cells activate a battery of checkpoint signaling cascades to control cell cycle progression and elicit DNA repair in order to maintain genomic stability and integrity. Identified as a homolog of its fission yeast counterpart, human Rad9 was proposed to form a Rad9-Hus1-Rad1 protein complex to mediate checkpoint signals. However, the precise function of Rad9 in the process of checkpoint activation is not fully understood. Using the RNA interference technique, we investigated the role of Rad9 in the genotoxic stress-induced activation of S-phase checkpoint and the maintenance of chromosomal stability. We found that Rad9 knockdown reduced the phosphorylation of Rad17, Chk1 and Smc1 in response to DNA replication block and certain types of DNA damage. Immunofluorescence studies showed that the removal of Rad9 disrupted the foci formation of phosphorylated Chk1, but not ATR. Moreover, Rad9 knockdown resulted in radioresistant DNA synthesis and reduced cell viability under replication stress. Finally, removal of Rad9 by RNAi led to increased accumulation of spontaneous chromosomal aberrations. Taken together, these results suggest a critical and specific role of Rad9 in the activation of S-phase checkpoint and the maintenance of chromosome stability.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2003

Human T-cell leukemia virus-I tax oncoprotein functionally targets a subnuclear complex involved in cellular DNA damage-response.

The virally encoded oncoprotein Tax has been implicated in HTLV-1-mediated cellular transformation. The exact mechanism by which this protein contributes to the oncogenic process is not known. However, it has been hypothesized that Tax induces genomic instability via repression of cellular DNA repair. We examined the effect of de novo Tax expression upon the cell cycle, because appropriate activation of cell cycle checkpoints is essential to a robust damage-repair response. Upon induction of tax expression, Jurkat T-cells displayed a pronounced accumulation in G2/M that was reversible by caffeine. We examined the G2-specific checkpoint signaling response in these cells and found activation of the ATM/chk2-mediated pathway, whereas the ATR/chk1-mediated response was unaffected. Immunoprecipitation with anti-chk2 antibody results in co-precipitation of Tax demonstrating a direct interaction of Tax with a chk2-containing complex. We also show that Tax targets a discrete nuclear site and co-localizes with chk2 and not chk1. This nuclear site, previously identified as Tax Speckled Structures (TSS), also contains the early damage response factor 53BP1. The recruitment of 53BP1 to TSS is dependent upon ATM signaling and requires expression of Tax. Specifically, Tax expression induces redistribution of diffuse nuclear 53BP1 to the TSS foci. Taken together these data suggest that the TSS describe a unique nuclear site involved in DNA damage recognition, repair response, and cell cycle checkpoint activation. We suggest that association of Tax with this multifunctional subnuclear site results in disruption of a subset of the site-specific activities and contributes to cellular genomic instability.

Carrier Proteins↗
PubMed2006

YC-1 induces S cell cycle arrest and apoptosis by activating checkpoint kinases.

Hypoxia-inducible factor-1alpha (HIF-1alpha) seems central to tumor growth and progression because it up-regulates genes essential for angiogenesis and the hypoxic adaptation of cancer cells, which is why HIF-1alpha inhibition is viewed as a cancer therapy strategy. Paradoxically, HIF-1alpha also leads to cell cycle arrest or the apoptosis of cancer cells. Thus, the possibility cannot be ruled out that HIF-1alpha inhibitors unlock cell cycle arrest under hypoxic conditions and prevent cell death, which would limit the anticancer effect of HIF-1alpha inhibitors. Previously, we reported on the development of YC-1 as an anticancer agent that inhibits HIF-1alpha. In the present study, we evaluated the effects of YC-1 on hypoxia-induced cell cycle arrest and cell death. It was found that YC-1 does not reverse the antiproliferative effect of hypoxia, but rather that it induces S-phase arrest and apoptosis at therapeutic concentrations that inhibit HIF-1alpha and tumor growth; however, YC-1 did not stimulate cyclic guanosine 3',5'-monophosphate production in this concentration range. It was also found that YC-1 activates the checkpoint kinase-mediated intra-S-phase checkpoint, independently of ataxia-telangiectasia mutated kinase or ataxia-telangiectasia mutated and Rad3-related kinase. These results imply that YC-1 does not promote the regrowth of hypoxic tumors because of its cell cycle arrest effect. Furthermore, YC-1 may induce the combined anticancer effects of HIF-1alpha inhibition and cell growth inhibition.

Apoptosis↗
PubMed2003

MDC1 is a mediator of the mammalian DNA damage checkpoint.

To counteract the continuous exposure of cells to agents that damage DNA, cells have evolved complex regulatory networks called checkpoints to sense DNA damage and coordinate DNA replication, cell-cycle arrest and DNA repair. It has recently been shown that the histone H2A variant H2AX specifically controls the recruitment of DNA repair proteins to the sites of DNA damage. Here we identify a novel BRCA1 carboxy-terminal (BRCT) and forkhead-associated (FHA) domain-containing protein, MDC1 (mediator of DNA damage checkpoint protein 1), which works with H2AX to promote recruitment of repair proteins to the sites of DNA breaks and which, in addition, controls damage-induced cell-cycle arrest checkpoints. MDC1 forms foci that co-localize extensively with gamma-H2AX foci within minutes after exposure to ionizing radiation. H2AX is required for MDC1 foci formation, and MDC1 forms complexes with phosphorylated H2AX. Furthermore, this interaction is phosphorylation dependent as peptides containing the phosphorylated site on H2AX bind MDC1 in a phosphorylation-dependent manner. We have shown by using small interfering RNA (siRNA) that cells lacking MDC1 are sensitive to ionizing radiation, and that MDC1 controls the formation of damage-induced 53BP1, BRCA1 and MRN foci, in part by promoting efficient H2AX phosphorylation. In addition, cells lacking MDC1 also fail to activate the intra-S phase and G2/M phase cell-cycle checkpoints properly after exposure to ionizing radiation, which was associated with an inability to regulate Chk1 properly. These results highlight a crucial role for MDC1 in mediating transduction of the DNA damage signal.

Adaptor Proteins, Signal Transducing↗
PubMed2004

ATM-dependent CHK2 activation induced by anticancer agent, irofulven.

Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114) is one of a new class of anticancer agents that are semisynthetic derivatives of the mushroom toxin illudin S. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor types. Mechanisms of action studies indicate that irofulven induces DNA damage, MAPK activation, and apoptosis. In this study we found that in ovarian cancer cells, CHK2 kinase is activated by irofulven while CHK1 kinase is not activated even when treated at higher concentrations of the drug. By using GM00847 human fibroblast expressing tetracycline-controlled, FLAG-tagged kinase-dead ATR (ATR.kd), it was demonstrated that ATR kinase does not play a major role in irofulven-induced CHK2 activation. Results from human fibroblasts proficient or deficient in ATM function (GM00637 and GM05849) indicated that CHK2 activation by irofulven is mediated by the upstream ATM kinase. Phosphorylation of ATM on Ser(1981), which is critical for kinase activation, was observed in ovarian cancer cell lines treated with irofulven. RNA interference results confirmed that CHK2 activation was inhibited after introducing siRNA for ATM. Finally, experiments done with human colon cancer cell line HCT116 and its isogenic CHK2 knockout derivative; and experiments done by expressing kinase-dead CHK2 in an ovarian cancer cell line demonstrated that CHK2 activation contributes to irofulven-induced S phase arrest. In addition, it was shown that NBS1, SMC1, and p53 were phosphorylated in an ATM-dependent manner, and p53 phosphorylation on serine 20 is dependent on CHK2 after irofulven treatment. In summary, we found that the anticancer agent, irofulven, activates the ATM-CHK2 DNA damage-signaling pathway, and CHK2 activation contributes to S phase cell cycle arrest induced by irofulven.

Antineoplastic Agents, Alkylating↗
PubMed2003

A DNA damage checkpoint response in telomere-initiated senescence.

Most human somatic cells can undergo only a limited number of population doublings in vitro. This exhaustion of proliferative potential, called senescence, can be triggered when telomeres--the ends of linear chromosomes-cannot fulfil their normal protective functions. Here we show that senescent human fibroblasts display molecular markers characteristic of cells bearing DNA double-strand breaks. These markers include nuclear foci of phosphorylated histone H2AX and their co-localization with DNA repair and DNA damage checkpoint factors such as 53BP1, MDC1 and NBS1. We also show that senescent cells contain activated forms of the DNA damage checkpoint kinases CHK1 and CHK2. Furthermore, by chromatin immunoprecipitation and whole-genome scanning approaches, we show that the chromosome ends of senescent cells directly contribute to the DNA damage response, and that uncapped telomeres directly associate with many, but not all, DNA damage response proteins. Finally, we show that inactivation of DNA damage checkpoint kinases in senescent cells can restore cell-cycle progression into S phase. Thus, we propose that telomere-initiated senescence reflects a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Both DNA topoisomerase II-binding protein 1 and BRCA1 regulate the G2-M cell cycle checkpoint.

Cell cycle checkpoints play a central role in genomic stability. The human DNA topoisomerase II-binding protein 1 (TopBP1) protein contains eight BRCA1 COOH terminus motifs and shares similarities with Cut5, a yeast checkpoint Rad protein. TopBP1 also shares many features with BRCA1. We report that, when expression of TopBP1 protein is inhibited in BRCA1 mutant cells, mimicking a TopBP1, BRCA1 double-negative condition, the G(2)-M checkpoint is strongly abrogated and apoptosis is increased after ionizing radiation. However, a BRCA1-negative or a TopBP1-negative background resulted in only partial abrogation of the G(2)-M checkpoint. The BRCA1 mutant and TopBP1-reduced condition specifically destroys regulation of the Chk1 kinase but not the Chk2 kinase, suggesting involvement in the ataxia telangiectasia-related pathway. These results indicate that both TopBP1 and BRCA1 specifically regulate the G(2)-M checkpoint, partially compensating each function.

Apoptosis↗
PubMed2005

N-methyl-N'-nitro-N-nitrosoguanidine activates cell-cycle arrest through distinct mechanisms activated in a dose-dependent manner.

S(N)1-alkylating agents, such as the mutagenic and cytotoxic drug N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), robustly activate the DNA damage-responsive G(2) checkpoint. Establishment of this checkpoint is dependent on a functional mismatch repair (MMR) system; however, exposure to high doses of MNNG overrides the requirement for MMR to trigger G(2) arrest. In addition, unlike moderate-dose exposure, in which the G(2) checkpoint is attenuated in ataxia-telangiectasia, mutated (ATM)-deficient cells, high-dose MNNG treatment activates G(2) arrest through an ATM-independent mechanism. We document that this arrest is sensitive to the pharmacological agents caffeine and 7-hydroxystaurosporine (UCN-01) that inhibit the checkpoint kinases ATM/ATM and Rad-3-related (ATR) and Chk1/Chk2, respectively. Furthermore, these agents block inactivation of the cell-cycle regulatory molecules Cdc25C and Cdc2, establishing the downstream mechanism through which high-dose MNNG establishes G(2) arrest. Activation of both Chk2 and Chk1 was independent of ATM and MMR in response to high-dose MNNG, unlike the response to moderate doses of this drug. Chk2 was found to be dispensable for cell-cycle arrest in response to high-dose MNNG treatment; however, ATR deficiency and decreased Chk1 expression forced by RNA interference resulted in diminished checkpoint response. These results indicate that MNNG activates the G(2) checkpoint through different mechanisms activated in a dose-dependent fashion.

Cell Cycle↗
PubMed2000

Analysis of fission yeast primase defines the checkpoint responses to aberrant S phase initiation.

To investigate the checkpoint response to aberrant initiation, we analyzed the cell cycle checkpoint response induced by mutations of Schizosaccharomyces pombe DNA primase. DNA primase has two subunits, Spp1 and Spp2 (S. pombe primases 1 and 2). Spp1 is the catalytic subunit that synthesizes the RNA primer, which is then extended by DNA polymerase alpha (Polalpha) to synthesize an initiation DNA structure, and this catalytic function of Polalpha is a prerequisite for generating the S-M phase checkpoint. Here we show that Spp2 is required for coupling the function of Spp1 to Polalpha. Thermosensitive mutations of spp2(+) destabilize the Polalpha-primase complex, resulting in an allele-specific S phase checkpoint defect. The mutant exhibiting a more severe checkpoint defect also has a higher extent of Polalpha-primase complex instability and deficiency in the hydroxyurea-induced Cds1-mediated intra-S phase checkpoint response. However, this mutant is able to activate the Cds1 response to S phase arrest induced by temperature. These findings suggest that the Cds1 response to the S-phase arrest signal(s) induced by a initiation mutant is different from that induced by hydroxyurea. Interestingly, a polalphats mutant with a defective S-M phase checkpoint and an spp2 mutant with an intact checkpoint have a similar Polalpha-primase complex stability, and the Cds1 response induced by hydroxyurea or by the mutant arrests at the restrictive temperature. Thus, the Cds1-mediated intra-S phase checkpoint response induced by hydroxyurea can also be distinguished from the S-M phase checkpoint response that requires the initiation DNA synthesis by Polalpha.

Alleles↗
PubMed2005

SQ/TQ cluster domains: concentrated ATM/ATR kinase phosphorylation site regions in DNA-damage-response proteins.

ATM/ATR-like protein kinases play central roles in the maintenance of genome stability and phosphorylate numerous substrates in response to DNA damage, preferentially on SQ or TQ motifs. ATM/ATR substrates often contain several closely spaced SQ/TQ motifs in regions that have been termed SQ/TQ cluster domains (SCDs). SCDs are now considered a structural hallmark of DNA-damage-response proteins. Mutational analyses of a number of SCD-containing proteins indicate that multisite phosphorylation of SQ/TQ motifs is required for normal DNA-damage responses, most commonly by mediating protein-protein interactions in the formation of DNA-damage-induced complexes. SCD sequences are highly diverse and these domains may be largely unfolded in their native state rather than adopting a common three-dimensional fold. Structural disorder of SCDs could be advantageous for efficient phosphorylation by ATM/ATR kinases and also enable them to be molded into distinct conformations to facilitate flexible interactions with multiple binding partners.

Amino Acid Motifs↗
PubMed2002

Increased expression of CHK2 in human gastric carcinomas harboring p53 mutations.

Human Chk1 and Chk2 are DNA damage-activated protein kinases that function as downstream mediators of ataxia-telangiectasia mutated (ATM), which is involved in G(2)/M cell cycle arrest. To clarify the relation between the expression of Chk1/Chk2 and p53 gene status in human gastric carcinomas, we examined expression of Chk1, Chk2 and p53 proteins in 87 gastric carcinomas by Western blotting and immunohistochemistry. We found a significant correlation between the expression levels of Chk1 and p53 proteins in gastric carcinomas (p = 0.016). Significant statistical association was also observed between levels of Chk2 and p53 proteins (p = 0.00024). To clarify the genetic alterations of p53 in gastric carcinomas, we performed PCR-SSCP analysis on 47 gastric carcinomas. Although we found that 5 of 7 (71%) gastric cancers expressed elevated levels of Chk1 had p53 mutation, there was not a statistically significant correlation between expression of Chk1 and genetic status of p53. We also found that 7 of 11 (78%) gastric carcinomas expressed elevated levels of Chk2 had p53 mutation, and this correlation was significant (p = 0.0157). We used a highly quantitative 5' nuclease fluorogenic RT-PCR method (TaqMan) to analyze the expression of Chk2 mRNA in 22 gastric carcinomas. Chk2 mRNA expression was higher in gastric carcinomas with p53 mutations compared to those harboring wild-type p53. A significant association was recognized between the expression of Chk2 mRNA and p53 mutational status (p = 0.031). Our findings support the hypothesis that expression of Chk2 protein is increased in gastric carcinomas with mutant p53. Chk1 and Chk2 may play important roles in the checkpoint function in human gastric carcinomas harboring p53 mutation when their functions are preserved to prevent cell cycle progression.

Adenocarcinoma, Papillary↗
PubMed2007

Centrosomal localization of DNA damage checkpoint proteins.

During mitosis, the phosphatidylinositol-3 (PI-3) family-related DNA damage checkpoint kinases ATM and ATR were found on the centrosomes of human cells. ATRIP, an interaction partner of ATR, as well as Chk1 and Chk2, the downstream targets of ATR or ATM, were also localized to the centrosomes. Surprisingly, the DNA-PK inhibitor vanillin enhanced the level of ATM on centrosomes. Accordingly, DNA-PKcs, the catalytic subunit of DNA-PK, was also found on the centrosomes. Vanillin altered the phosphorylation of Chk2 in the centrosomes and in whole cell extracts. Nucleoplasmic ATM co-immunoprecipitated with Ku70/86, the DNA binding subunits of DNA-PK, while vanillin diminished this association. Vanillin did not affect microtubule polymerization at the centrosomes but, surprisingly, caused a transient enhancement of alpha-tubulin foci in the nucleus. Interestingly, gamma-tubulin was also present in the nucleus and co-immunoprecipitated with ATR or BRCA1. DNA damage led to a reduction of the mentioned checkpoint proteins on the centrosomes but increased the level of gamma-tubulin at this organelle. Taken together, these results indicate that DNA damage checkpoint proteins may control the formation of gamma-tubulin and/or the kinetics of microtubule formation at the centrosomes, and thereby couple them to the DNA damage response.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, which is a structural homolog of the RAD2 and RAD27 DNA repair genes.

A number of DNA damage-inducible genes (DIN) have been identified in Saccharomyces cerevisiae. In the present study we describe isolation of a novel gene, Din7, the expression of which is induced by exposure of cells to UV light, MMS (methyl methane-sulfonate) or HU (hydoxyurea). The DNA sequence of DIN7 was determined. By comparison of the predicted Din7 amino acid sequence with those in databases we found that it belongs to a family of proteins which includes S. cerevisiae Rad2 and its Schizosaccharomyces pombe and human homologs Rad13 and XPGC; S. cerevisiae Rad27 and its S. pombe homolog Rad2, and S. pombe Exo I. All these proteins are endowed with DNA nuclease activity and are known to play an important function in DNA repair. The strongest homology to Din7 was found with the Dhs1 protein of S. cerevisiae, the function of which is essentially unknown. The expression of the DIN7 gene was studied in detail using a DIN7-lacZ fusion integrated into a chromosome. We show that the expression level of DIN7 rises during meiosis at a time nearly coincident with commitment to recombination. No inducibility of DIN7 was found after treatment with DNA-damaging agents of cells bearing the rad53-21 mutation. Surprisingly, a high basal level of DIN7 expression was found in strains in which the DUN1 gene was inactivated by transposon insertion. We suggest that a form of Dun1 may be a negative regulator of the DIN7 gene expression.

Amino Acid Sequence↗
PubMed1999

The S/M checkpoint at 37 degrees C and the recovery of viability of the mutant poldeltats3 require the crb2+/rhp9+ gene in fission yeast.

We have isolated a mutant in fission yeast, in which mitosis is uncoupled from completion of DNA replication when DNA synthesis is impaired by a thermosensitive mutation in the gene encoding the catalytic subunit of DNA polymerase delta. By functional complementation, we cloned the wild-type gene and identified it as the recently cloned checkpoint gene crb2+/rhp9+. This gene has been implicated in the DNA damage checkpoint and acts in the Chk1 pathway. Unlike the deleted strain dcrb2, cells bearing the crb2-1 allele were not affected in the DNA repair checkpoint after UV or MMS treatment at 30 degrees C, but were defective in this checkpoint function when treated with MMS at 37 degrees C. We analysed the involvement of Crb2 in the S/M checkpoint by blocking DNA replication with hydroxyurea, by using S phase cdc mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37 degrees C. Furthermore, the crb2+ gene was required, together with the cds1+ gene, for the S/M checkpoint at 30 degrees C. Finally, both the crb2 deletion and the crb2-1 allele induced a rapid death phenotype in the poldeltats3 background at both 30 degrees C and 37 degrees C. The rapid death phenotype was independent of the checkpoint functions.

Cell Cycle Proteins↗