Resume Cell Cycle Progression Research Articles

Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion-induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.

Transcription factor-mediated epigenetic regulation of cell growth and phenotype for biological control and cancer

There is a requirement to retain regulatory information and parameters of nuclear organization during cell division when transcription is globally silenced to sustain competency for expression of genes that control proliferation, cell growth and phenotype in progeny cells. Histone modifications, DNA methylation and RNA-mediated silencing are well defined, DNA-independent epigenetic mechanisms that regulate gene expression. Recent results suggest that retention of lineage-specific transcription factors with promoter elements and sequential assembly of gene regulatory machinery is a novel parameter of epigenetic control to sustain cellular identity post-mitotically. Collectively, these epigenetic signatures “bookmark” genes for activation or suppression as cells exit mitosis and resume cell cycle progression. We propose a model for cell fate and lineage commitment, coordination and maintenance where the regulatory proteins are retained on mitotic chromosomes to convey information for biological control to progeny cells as well as for controlling expression of the transformed and tumor phenotypes. MicroRNAs are another dimension to transcription factor-mediated epigenetic regulation of gene expression in biological control and cancer. Our findings demonstrate that fidelity of transcription factor localization is required for microRNA-related activation and suppression of regulatory networks in myeloid progenitor cells providing a novel and selective epigenetic target for treatment of acute myelogenous leukemia.

Cellular senescence in human mammary epithelial cells (HMEC) is accompanied by an increase of elastin formation

Normal human mammary epithelial cells (HMEC) have a finite life span, regulated by a process termed cellular senescence. After an initial stress-associated senescence barrier, some cells are subjected to a self-selection process triggered by the loss of the G1 cell cycle regulator p16INK4a expression. This effect is associated with an escape from the growth plateau to resume cell cycle progression and to undergo further cell divisions. Thereafter, these so-called HMEC encounter a second senescence barrier, termed agonescence. Our recent work using an siRNA approach revealed an essential impact of the extracellular matrix metalloproteinase-7 (MMP-7) on the aging process of post-selection HMEC. Since MMP-7 is involved in a variety of cellular mechanisms, including processing of inactive precursor proteins, ectodomain shedding of extracellular signaling molecules and remodeling of the extracellular matrix (ECM), respectively, we could observe considerable alterations of the ECM during senescence of HMEC. Thus, the formation of characteristic structures similar to elastin was detectable, which markedly increased in agonescent HMEC cultures after passage 15. Moreover, Western blot analysis revealed an up-regulated expression of tropoelastin, the soluble precursor of the ECM protein elastin, during the aging process of HMEC. This was paralleled by an enhanced activity of the enzyme lysyl oxidase (LOX) which is responsible for cross-linking tropoelastin monomers to form insoluble elastic fibers. Thus, enhanced LOX activity was detectable in cellular homogenates as well as in the culture supernatant during HMEC aging between passage 12 up to passage 16. Together with the down-regulation of MMP-7, the increased formation of elastic fibers supported an essential effect of distinct ECM components and the microenvironment on HMEC aging [1].

Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete

In response to a DNA double-strand break (DSB), chromatin is rapidly modified by the damage dependent checkpoint kinases. Also, disassembly of chromatin occurs at the break site. The damage-induced modification of chromatin structure is involved in the maintenance of the checkpoint. However, it has not been determined how chromatin is restored to its undamaged state when DSB repair is complete. Here, we show the involvement of two chromatin assembly factors (CAFs), Asf1 and CAF-1, in turning off the DNA damage checkpoint in budding yeast. DSB repair or formation of gamma-H2AX does not depend on either the CAF-1 protein, Cac1, or Asf1. Absence of these proteins does not impair the ability of cells to resume cell cycle progression in the presence of an unrepaired DSB (adaptation). However, recovery from cell cycle checkpoint arrest when the DSB is repaired by gene conversion is substantially defective in the absence of both CAF-1 and Asf1, whereas deleting CAC1 or ASF1 individually had little effect. We suggest that CAF-1 and Asf1 function redundantly to deactivate the checkpoint by restoring chromatin structure on the completion of DSB repair.

The polo-like kinase 1 regulates CDC25B-dependent mitosis entry

Activation of cyclin-dependent kinase complexes (CDK) at key cell cycle transitions is dependent on their dephosphorylation by CDC25 dual-specificity phosphatases (CDC25A, B and C in human). The CDC25B phosphatase plays an essential role in controlling the activity of CDK1-cyclin B complexes at the entry into mitosis and together with polo-like kinase 1 (PLK1) in regulating the resumption of cell cycle progression after DNA damage-dependent checkpoint arrest in G2. In this study, we analysed the regulation of CDC25B-dependent mitosis entry by PLK1. We demonstrate that PLK1 activity is essential for the relocation of CDC25B from the cytoplasm to the nucleus. By gain and loss of function analyses, we show that PLK1 stimulates CDC25B-induced mitotic entry in both normal conditions and after DNA-damage induced G2/M arrest. Our results support a model in which the relocalisation of CDC25B to the nucleus at the G2-M transition by PLK1 regulates its mitotic inducing activity.

Transient suppression of nuclear Cdc2 activity in response to ionizing radiation

Suppression of Cdc2 activity is essential for DNA damage-induced G2 arrest. In the present study, we elucidated regulatory mechanism of Cdc2 activity during radiation-induced transient G2 arrest. Exposure of the cells to gamma-radiation (4 Gy) led to a transient increase of cells in G2 at 12 h rather than M phase and then the cells resumed cell cycle progression from the G2 arrest. However, the levels of cyclin B1 and Cdc2 activity were increased in the whole cell extracts at 12 h. Despite cyclin B1 induction and increased level of Cdc2 activity after irradiation the activities in the nuclear fractions were transiently decreased at 12 h and returned to control levels by 24-48 h, demonstrating transient inhibition of nuclear translocation of cyclin B1 in response to radiation. Moreover, inhibitory phosphorylation of the Cdc2 on Tyr15 and the Cdc25C on Ser216 were increased concomitant with transient G2 arrest. The level of phosphorylated Wee1 and its activity were also markedly increased at 12 h after irradiation. In addition, radiation caused nuclear accumulation of p21(CIP1/WAF1) at 12 h, resulting in increased-binding of p21(CIP1/WAF1) to Cdc2. Nuclear p21(CIP1/WAF1) protein level and its binding to Cdc2 gradually returned to control level when the cells resumed cell cycle progression. However, total protein level of p21(CIP1/WAF1) continued to increase until 48 h after irradiation. Collectively, these results indicate that the suppression of nuclear import of cyclin B1, the induction of Wee1 kinase activity, and the transient nuclear accumulation of p21(CIP1/WAF1) may play important roles in the transient cell cycle delay in response to ionizing radiation.

RNA interference targeting ORC1 gene suppresses the proliferation of vascular smooth muscle cells in rats

Background The proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. The largest subunit of the origin recognition complex (ORC), ORC1, plays a critical role during the initiation of DNA replication in eukaryotes. However, the involvement of ORC1 in the initiation of DNA replication in VSMCs has not been studied yet. Objective The aim of this study was to silence ORC1 gene selectively by using RNA interference and analyze the effects of ORC1 gene on the proliferation and apoptosis of rat VSMCs. Methods Freshly isolated rat VSMCs were transfected with siRNA targeting ORC1 gene capsulated in liposome. ORC1 protein expression was determined by Western blotting and ORC1 mRNA level by RT-PCR. DNA synthesis was analyzed by 3H thymidine ( 3H-TdR) incorporation and cell proliferative activity and cell cycle distribution by flow cytometry. Two apoptosis-related proteins, Bax and Bcl-2, were examined immunohistochemically. Results Down-regulation of ORC1 mRNA and protein expression was observed in rat VSMCs at 24 h after transfection with the three pairs of siRNA targeting ORC1 gene and this reduction persisted at least 7 days post-transfection. Down-regulation of ORC1 mRNA (60%) and protein (80%) expression was observed at 72 h post-transfection in the cells transfected with B-ORC1 siRNA. A significant decrease in 3H thymidine incorporation was observed in rat VSMCs with ORC1 gene silencing after serum challenge, but not in the non-silenced control. A significant increase in the proliferation index and a significant decrease in the percentage of cells at G 0/G 1 phase after serum challenge were observed in the non-silenced control, but not in ORC1 gene silenced cells. A significant increase in the ratio of Bcl-2/Bax was observed after serum challenge in the non-silenced control, but only a slight increase was found in the ORC1 gene silenced cells. ORC1 gene silencing disappeared 7 days after transfection. Continuous serum challenge stimulated VSMCs to synchronously reenter the cell cycle as evidenced by increases in [ 3H] thymidine incorporation, the proliferation index, and the ratio of Bcl-2/Bax, as non-silenced cells were induced to resume cell cycle progression by the addition of 15% fetal bovine serum to the culture medium. Conclusion ORC1 gene silencing causes rat VSMCs to enter a reversible G 0 quiescent, growth arrested state; thus, ORC1 gene may be an important new target for suppressing VSMCs proliferation.

Mismatch Repair Status and the Response of Human Cells to Cisplatin

The emergence of resistance to cisplatin is a serious drawback of cancer therapy. To help elucidate the molecular basis of this resistance, we examined matched ovarian cancer cell lines that differ in their DNA mismatch repair (MMR) status and the response to cisplatin. Checkpoint activation by cisplatin was identical in both lines. However, sensitive cells delayed S-phase transition, arrested at G2/M and died by apoptosis. The G2/M block was characterized by selective disappearance of homologous recombination (HR) proteins, which likely resulted in incomplete repair of the cisplatin adducts. In contrast, resistant cells transiently arrested at G2/M, maintained constant levels of HR proteins and ultimately resumed cell cycle progression. The net contribution of MMR to the cisplatin response was examined using matched semi-isogenic (HCT116±chr3) or strictly isogenic (293T-Lα-/+) cell lines. Delayed transition through S-phase in response to cisplatin was also observed in the MMR-proficient HCT116+chr3 cells. Unlike in the ovarian cell lines, however, both HCT116+chr3 and HCT116 permanently arrested at G2/M with an intact complement of HR proteins and died by apoptosis. A similar G2/M arrest was observed in the strictly isogenic 293T-Lα-/+ cells. This confirmed that although MMR undoubtedly contributes towards the cytotoxicity of cisplatin, it is only one of several pathways that modulate the cellular response to this drug. However, our data highlighted the importance of HR to cisplatin cytotoxicity and suggested that HR status might represent a novel prognostic marker and possibly also a therapeutic target, the inhibition of which would substantially sensitize cells to cisplatin chemotherapy.

The yeast DNA damage checkpoint proteins control a cytoplasmic response to DNA damage

A single HO endonuclease-induced double-strand break (DSB) is sufficient to activate the DNA damage checkpoint and cause Saccharomyces cells to arrest at G(2)/M for 12-14 h, after which cells adapt to the presence of the DSB and resume cell cycle progression. The checkpoint signal leading to G(2)/M arrest was previously shown to be nuclear-limited. Cells lacking ATR-like Mec1 exhibit no DSB-induced cell cycle delay; however, cells lacking Mec1's downstream protein kinase targets, Rad53 or Chk1, still have substantial G(2)/M delay, as do cells lacking securin, Pds1. This delay is eliminated only in the triple mutant chk1Delta rad53Delta pds1Delta, suggesting that Rad53 and Chk1 control targets other than the stability of securin in enforcing checkpoint-mediated cell cycle arrest. The G(2)/M arrest in rad53Delta and chk1Delta revealed a unique cytoplasmic phenotype in which there are frequent dynein-dependent excursions of the nucleus through the bud neck, without entering anaphase. Such excursions are infrequent in wild-type arrested cells, but have been observed in cells defective in mitotic exit, including the semidominant cdc5-ad mutation. We suggest that Mec1-dependent checkpoint signaling through Rad53 and Chk1 includes the repression of nuclear movements that are normally associated with the execution of anaphase.

Evidence for the involvement of purα in response to DNA replication stress

Purα is a sequence-specific nucleic acid binding protein that is involved in multiple cellular functions including regulation of transcription, initiation of DNA replication, cell cycle progression, and neuronal cell differentiation. Its potential role in DNA repair has not been explored. We have now analyzed the role of Purα in the cellular response to replication-associated DNA repair of double-strand breaks (DSBs) using Purα knockout mouse embryo fibroblasts (MEFs). We found that Purα negative cells are hypersensitive to the DNA replication inhibitor, hydroxyurea (HU), and that HU induces excessive DSBs, which delayed the resumption of cell cycle progression after HU treatment. Reintroduction of Purα into Purα null cells reduced the accumulation of DSBs and enhanced DNA end joining. These results suggest a role for Purα as a caretaker protein that is involved in the repair of DSBs induced by stalled replication forks.

Induction of Cdc25B Regulates Cell Cycle Resumption after Genotoxic Stress

Cdc25 phosphatases propel cell cycle progression by activating cyclin-dependent kinases (Cdk). DNA damage is generally thought to inhibit Cdc25 functionality by inducing proteasomal degradation of Cdc25A and phosphorylation-mediated sequestration of Cdc25B and Cdc25C to the cytoplasm. More recently, a critical role for Cdc25B in the resumption of cell cycle progression through mitosis after DNA damage has been identified. In this study, the fate of Cdc25B after mechanistically distinct DNA-damaging agents (etoposide, cisplatin, bleomycin, ionizing irradiation, or UV irradiation) was examined, and surprisingly a rapid increase in cellular Cdc25B levels was observed after DNA damage. Using UV irradiation as the prototypic damaging agent, we found that the increase in Cdc25B levels was checkpoint dependent and was controlled by a p53-independent mechanism. Cdc25B levels controlled the number of cells progressing into mitosis after UV, but they did not affect G(2)-M checkpoint engagement immediately after DNA damage. Increased Cdc25B reduced the time required for cell cycle resumption. These data support a model in which Cdc25B accumulation is an important anticipatory event for cell cycle resumption after DNA damage.

Telomerase- and recombination-independent immortalization of budding yeast.

It is generally assumed that there are only two ways to maintain the ends of chromosomes in yeast and mammalian nuclei: telomerase and recombination. Without telomerase and recombination, cells enter senescence, a state of permanent growth arrest. We found that the decisive role in preventing senescent budding yeast cells from dividing is played by the Exo1 nuclease. In the absence of Exo1, telomerase- and recombination-defective yeast can resume cell cycle progression, despite degradation of telomeric regions from many chromosomes. As degradation progresses toward internal chromosomal regions, a progressive decrease in viability would be expected, caused by loss of essential genes. However, this was not the case. We demonstrate that extensive degradation and loss of essential genes can be efficiently prevented through a little-studied mechanism of DNA double-strand-break repair, in which short DNA palindromes induce formation of large DNA palindromes. For the first time, we show that large palindromes form as a natural consequence of postsenescence growth and that they become essential for immortalization in the absence of telomerase activity.

Involvement of base excision repair in response to therapy targeted at thymidylate synthase

Thymidylate synthase (TS) is an important target of several classes of chemotherapeutic agents. Although the precise mechanism of cytotoxicity in thymidylate deprivation remains obscure, uracil misincorporation and DNA strand breaks are recognized as important events during thymidylate deprivation. Base excision repair (BER) plays a primary role in removing damaged or modified bases from the genome, including uracil. Because of uracil misincorporation, BER is hypothesized to play a role in the cellular response to thymidylate deprivation. In this study, we used murine embryo fibroblasts wild-type or homozygous null for DNA polymerase beta (beta-pol), which plays a central role in BER. We found that, compared with wild-type, beta-pol null cells were resistant to the toxic effects of raltitrexed (Tomudex, ZD1694), a folate inhibitor of TS. There was little difference in TS levels or in TS-ligand complex formation between the cell lines. Furthermore, cells deficient in XRCC1, a scaffold protein for the final steps of BER, were also modestly resistant to raltitrexed compared with XRCC1-proficient cells. Cell cycle analysis revealed that the responses of the wild-type and beta-pol null cells were similar during drug exposure. However, following drug removal, the beta-pol null cells appeared to resume cell cycle progression more rapidly than the wild-type cells. The results suggest that BER plays a role in modulating the toxic effects of TS inhibitors, and that this role occurs during recovery from TS inhibition.

Mechanisms involved in regulating DNA replication origins during the cell cycle and in response to DNA damage.

Replication origins in eukaryotic cells never fire more than once in a given S phase. Here, we summarize the role of cyclin-dependent kinases in limiting DNA replication origin usage to once per cell cycle in the budding yeast Saccharomyces cerevisiae. We have examined the role of different cyclins in the phosphorylation and regulation of several replication/regulatory factors including Cdc6, Sic1, ORC and DNA polymerase alpha-primase. In addition to being regulated by the cell cycle machinery, replication origins are also regulated by the genome integrity checkpoint kinases, Mec1 and Rad53. In response to DNA damage or drugs which interfere with the progression of replication forks, the activation of late-firing replication origins is inhibited. There is evidence indicating that the temporal programme of origin firing depends upon the local histone acetylation state. We have attempted to test the possibility that checkpoint regulation of late-origin firing operates through the regulation of the acetylation state. We found that overexpression of the essential histone acetylase, Esal, cannot override checkpoint regulation of origin firing. We have also constructed a temperature-sensitive esa1 mutant. This mutant is unable to resume cell cycle progression after alpha-factor arrest. This can be overcome by overexpression of the G1 cyclin, Cln2, revealing a novel role for Esal in regulating Start.

A signal transduction protein cues proteolytic events critical to Caulobacter cell cycle progression.

Temporally controlled proteolysis of the essential response regulator, CtrA, is critical for cell cycle progression in Caulobacter crescentus. CtrA binds to and silences the origin of replication in swarmer cells. The initiation of replication depends on the proteolysis of CtrA. We present evidence that DivK, an essential single-domain response regulator, contributes to the control of the G(1)-S transition by signaling the temporally controlled proteolysis of CtrA. In a divK-cs mutant at the restrictive temperature, the initiation of DNA replication is blocked because of the retention of CtrA. A shift of cells from restrictive to permissive temperature results in rapid degradation of CtrA, initiation of DNA replication, and the resumption of cell cycle progression, including the ordered expression of genes involved in chromosome replication and polar organelle biogenesis. CtrA binds to and regulates the promoters of two genes critical to its temporally controlled proteolysis, divK and clpP, providing a transcriptional feedback loop for the control of cell cycle progression.

The Saccharomyces recombination protein Tid1p is required for adaptation from G2/M arrest induced by a double-strand break

Saccharomyces cells with a single unrepaired double-strand break (DSB) will adapt to checkpoint-mediated G2/M arrest and resume cell cycle progression. The decision to adapt is finely regulated by the extent of single-stranded DNA generated from a DSB. We show that cells lacking the recombination protein Tid1p are unable to adapt, but that this defect is distinct from any role in recombination. As with the adaptation-defective mutations yku70Δ and cdc5-ad, permanent arrest in tid1Δ is bypassed by the deletion of the checkpoint gene RAD9. Permanent arrest of tid1Δ cells is suppressed by the rfa1-t11 mutation in the ssDNA binding complex RPA, similar to yku70Δ, whereas the defect in cdc5-ad is not suppressed. Unlike yku70Δ, tid1Δ does not affect 5′-to-3′ degradation of DSB ends. The tid1Δ defect cannot be complemented by overexpressing the homolog Rad54p, nor is it affected in rad51Δ tid1Δ, rad54Δ tid1Δ, or rad52Δ tid1Δ double mutants that prevent essentially all homologous recombination. We suggest that Tid1p participates in monitoring the extent of single-stranded DNA produced by resection of DNA ends in a fashion that is distinct from its role in recombination.

Cell cycle progression and radiation survival following prolonged hypoxia and re-oxygenation

Purpose : To investigate cell cycle progression and radiation survival following prolonged hypoxia and re-oxygenation. Materials and methods : NHIK 3025 human cervical carcinoma cells were exposed to extremely hypoxic conditions (<4ppm O 2) for 20h and then re-oxygenated. The subsequent cell cycle progression was monitored by analysing cell cycle distribution at different time-points after re-oxygenation using two-dimensional flowcytometry. The clonogenic survival after a 3.6Gy X-ray dose was also measured at each of these time-points. The measured radiation survival was compared with theoretical predictions based on cell cycle distribution and the radiation age-response of the cells. Results : Following re-oxygenation the cells resumed cell cycle progression, completed S-phase, and then accumulated in G 2. Non-clonogenic cells remained permanently arrested in G 2, while the remainder of the cells completed mitosis after a few hours delay. The radiation survival of the hypoxia-pretreated cell population remained lower than for an exponentially growing control population for the investigated 50h of re-oxygenation. However, following 7h of re-oxygenation, the radiation survival of the hypoxia-treated cell population correlated well with theoretically predicted values based on cell cycle distribution and radiation age-response. Conclusions : The work demonstrates that prolonged hypoxia followed by re-oxygenation results in a G 2 delay similar to that observed after DNA damage. Furthermore, chronic hypoxia results in decreased radiation survival for at least 50h following the reintroduction of oxygen. The hypoxia-induced radiosensitization following 7h of re-oxygenation could in large part be explained by the synchronous cell cycle progression that occurred.

Heat shock effects on cell cycle progression.

In mammalian cells, short-term (acute) exposure to a moderate heat shock leads to a transient arrest of cells at mainly two cell cycle checkpoints, the G1/S and G2/M transitions. This is documented by the more or less synchronous resumption of cell cycle progression from these checkpoints during recovery. The reason for the accumulation of cells at these checkpoints may be found in activity thresholds of cyclin-dependent kinases (Cdks) at both transitions which are determined by (i) the amounts of the responsible cyclins, (ii) regulatory phosphorylation of the Cdks and (iii) the inhibition of Cdks by associated regulatory proteins (Ckis). All three regulatory systems may be subject to heat-shock-dependent changes, the amounts of Ckis, in particular, being increased. Cdk-dependent phosphorylation of the retinoblastoma protein and the subsequent release of active S-phase-specific transcription factors E2F/DP are considered as major heat-sensitive steps in cell cycle progression. Furthermore, high acute heat shock and long-term (chronic) heat treatment may lead to cell-type-specific forms of cell death. All types of responses to heat treatment are subject to adaptation after a 'priming' treatment, probably due to higher levels of heat shock proteins.

P34Cdc2 Kinase Activity Is Excluded from the Nucleus during the Radiation-induced G2 Arrest in HeLa Cells

The progression of cells from G(2) into mitosis is blocked by exposure to DNA-damaging agents such as ionizing radiation. This G(2) delay is associated with reduced cyclin B1-specific associated histone H1 kinase activity, increased inhibitory phosphorylation of p34(Cdc2), and depressed cyclin B1 levels in HeLa cells. Induction of cyclin B1 or expression of Cdc2AF, a mutant p34(Cdc2) that lacks the sites of inhibitory phosphorylation, only partially reverses the radiation-associated G(2) delay, although both maneuvers rapidly result in increased histone H1 kinase activity. To account for the persistent G(2) delay in the face of active p34(Cdc2) kinase, we determined the location of the kinase activity. Although p34(Cdc2) was active in the cytoplasm, the nuclear p34(Cdc2) was inactive. Irradiation led to nuclear accumulation of the inactive tyrosine-phosphorylated form of p34(Cdc2), whereas the active form was seen in the cytoplasm. At later times when cells had resumed cell cycle progression, nuclear kinase activity was detectable. These results give evidence of segregation of cytoplasmic and nuclear kinase activity after DNA damage that has the effect of enhancing checkpoint control. Shielding the nucleus from the potentially deleterious effects of kinase activity after DNA damage may help irradiated human cancer cells respond to irradiation.

Heat-induced cell cycle arrest of Saccharomyces cerevisiae: involvement of the RAD6/UBC2 and WSC2 genes in its reversal.

The Saccharomyces cerevisiae RAD6 (UBC2 ) gene encodes a ubiquitin-conjugating enzyme that is involved in a wide range of cellular processes including DNA repair, sporulation and N-end rule protein degradation. Under mild heat stress conditions (37-38 degrees C) rad6 null and rad6-149 mutant cells are unable to grow. The molecular basis for this failure to grow is unknown. Here we show that the heat sensitivity of rad6 mutants is not due to cell death but to an inability to progress in the cell cycle. The temperature-induced cell cycle arrest of these mutants is due to a block in a branch of the RAD6 pathway distinct from the DNA repair and the N-end rule protein degradation pathways. Wild-type cells heated to 38 degrees C arrest transiently in the late G1 phase and then resume growth. At 38 degrees C rad6 mutant cells arrest in late G1 but, unlike wild-type cells, are unable to resume cell cycle progression. In both wild-type and in rad6 mutant cells, CLN1 and CLN2 transcript levels fall sharply upon temperature increase. In wild-type cells levels of these transcripts recover rapidly, whereas in the rad6 mutant they recover slowly. As rad6 cells remain arrested even after CLN1 and CLN2 mRNAs regain their preheat stress levels, factors additional to reduced G1 cyclin gene expression must cause the temperature-induced cell cycle block of the mutant. To identify genes involved in the relief of the cell cycle arrest under heat stress, we screened a multicopy yeast genomic library for clones that restore the growth of the rad6-149 mutant. A plasmid was isolated carrying the WSC2 gene, which is closely related to WSC1/SLG1/HCS77, a putative membrane heat sensor. Overexpression of WSC2 reverses the heat-induced cell cycle arrest of rad6-149 but not of rad6 null mutants. Taken together the findings point to the existence of an unidentified heat stress-activated cell cycle checkpoint pathway, which is antagonized by Rad6p by a mechanism also involving Wsc2p.