Initiation Of S-phase Research Articles

The Human Licensing Factor for DNA Replication Cdt1 Accumulates in G1 and Is Destabilized after Initiation of S-phase

S-phase onset is controlled, so that it occurs only once every cell cycle. DNA is licensed for replication after mitosis in G(1), and passage through S-phase removes the license to replicate. In fission yeast, Cdc6/18 and Cdt1, two factors required for licensing, are central to ensuring that replication occurs once per cell cycle. We show that the human Cdt1 homologue (hCdt1), a nuclear protein, is present only during G(1). After S-phase onset, hCdt1 levels decrease, and it is hardly detected in cells in early S-phase or G(2). hCdt1 can associate with the DNA replication inhibitor Geminin, however these two proteins are mostly expressed at different cell cycle stages. hCdt1 mRNA, in contrast to hCdt1 protein, is expressed in S-phase-arrested cells, and its levels do not change dramatically during a cell cycle, suggesting that proteolytic rather than transcriptional controls ensure the timely accumulation of hCdt1. Consistent with this view, proteasome inhibitors stabilize hCdt1 in S-phase. In contrast, hCdc6/18 levels are constant through most of the cell cycle and are only low for a brief period at the end of mitosis. These results suggest that the presence of active hCdt1 may be crucial for determining when licensing is legitimate in human cells.

Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing.

The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures. Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content. Cdc40p is also required for spindle establishment or maintenance. Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing. In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms. Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore. Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export.

Regulation of the cell cycle by CDK inhibitors.

There are at least four possible ways in which inhibitors of cyclin-dependent kinases (CDKs) might regulate cell cycle progression (Fig. 1). First, they might determine the duration of G1 phase by setting a threshold above which G1 CDK activity must accumulate before initiation of S-phase. In this threshold role, steady-state levels of CDK inhibitors (CKIs) counteract the temporal accumulation of G1 cyclin/CDK complexes. Second, they might act as checkpoints inducing cell cycle arrest transiently when genomic fidelity is threatened. Chromosome damage or inappropriate segregation of chromosomes may induce cell cycle arrest and initiate DNA repair mechanisms or apoptotic programs. Third, CKIs might allow expedient withdrawal of cells from the cell cycle and a concomitant change in cell fate. In some cases, CKIs might not only regulate the withdrawal from the cell cycle but also affect lineage-specific functions. Fourth, they might function as a gatekeeper of quiescence, keeping the GO cell from inappropriately resuming proliferation. In some cases, tumors arise from well differentiated and non-mitotic cells that inexplicably return to the cell cycle. Over the last 5 years, biochemical, cellular, and molecular analyses of CKIs have combined with genetic analysis of mutant mice deficient in each CKI to address these possible functions. Our current knowledge of the role of CKI in these decisions and speculations about their roles in developmentally regulated cell cycles are discussed below.

A novel Mcm1-dependent element in the SWI4, CLN3, CDC6, and CDC47 promoters activates M/G1-specific transcription.

We have identified a novel promoter element that confers M/G1-specific transcription in Saccharomyces cerevisiae. This element, which we call an ECB (early cell cycle box), was first identified in the SWI4 promoter, but it is also present in the promoter of a G1 cyclin CLN3, as well as in the promoters of three DNA replication genes: CDC6, CDC47, and CDC46. Transcripts from all five of these genes oscillate during the cell cycle and peak at the M/G1 boundary, as do isolated ECB elements in reporter constructs. The ECB element contains an Mcm1 binding site to which Mcm1 binds in vitro, and an Mcm1-VP16 fusion, which places a constitutive activator on Mcm1-binding sites in vivo, can deregulate ECB-containing promoters. Mcm1 is a transcription factor that is also required for minichromosome maintenance. We provide evidence that the replication defect of mcm1 mutants can be suppressed by ectopic CDC6 transcription. Periodic expression of SWI4 and CLN3 may be important for cell cycle progression, as we find that these genes are both haploinsufficient and rate limiting for G1 progression. We suggest that ECB-regulated gene products play critical roles in promoting the initiation of S-phase, both by regulating CLN1 and CLN2 transcription and as components of the initiation complex on origins of replication.

The function of the Drosophila thioredoxin homologue encoded by the deadhead gene is redox-dependent and blocks the initiation of development but not DNA synthesis

Thioredoxin is a highly conserved disulfide reducing protein whose structure and biochemical properties have been extensively studied. Nonetheless, its function in vivo is not well defined. In Drosophila, the maternal-effect gene deadhead encodes a thioredoxin-like protein that is required for initiation of embryonic development. Here we report that deadhead function is dependent on its enzymatic activity: transgenes carrying mutations in thioredoxin's conserved active site failed to rescue the deadhead mutant phenotype. A number of studies have documented that thioredoxin plays a role in DNA synthesis. If thioredoxin is required for DNA synthesis in the fly, then deadhead mutations will suppress mutations that inappropriately synthesize DNA. Contrary to expectation, we find that deadhead does not function as a suppressor in this assay. The observed epistatic relationship between these mutations clearly indicates that deadhead is not essential for DNA metabolism. The possibility of a regulatory role in controlling the initiation of S-phase is discussed.

644 The cell cycle, checkpoints, and cancer

Studies carried out initially in yeast and marine invertebrate and amphibian eggs, have identified the cyclin dependent kinases (CDKs) as key elements in controlling the cell cycle. In the fission yeast a single CDK p34^cdc2 encoded by ^cdc2 is required to initiate both S-phase and mitosis, the major events common to all eukaryotic cell cycles. This protein kinase is regulated by several mechanisms, including the availability of B-cyclin to form an active complex with p34^cdc2, an inhibitory tyro-sine phosphorylation in the active site of the enzyme, and the action of a specific protein inhibitor. These mechanisms ensure that the p34^cdc2 protein kinase activity is precisely regulated during the cell cycle. Low level activity in G1 is required to bring about S-phase and during G2 to prevent a further round S-phase. Activation to high level in G2 initiates mitosis, and a failure to complete S-phase prevents this activation. Thus p34^cdc2 controls the onset of S-phase and mitosis and ensures they occur in the correct order during the cell cycle. The human <i>CDC2</i> gene was isolated by its ability to substitute for the <i>cdc2</i> gene in fission yeast. It controls the onset of mitosis in human cells indicating that the basic elements of cell cycle control are likely to be conserved in all eukaryotes. Other related CDKs have also been identified in human cells which act in G1, both in mid-G1 and at the onset of S-phase in late G1. These CDKs and their associated regulatory molecules play important roles in controlling cell cycle progression, and so have provided a new set of potential targets for restraining uncontrolled proliferation during malignancy. The CDKs are also important in the checkpoint controls which prevent initiation of S-phase and mitosis if earlier events of the cell cycle have not been properly completed. For example, premature p34^cdc2 activation can lead to mitosis without S-phase and inhibition of p34^cdc2 can lead to S-phase without mitosis. Defects of this sort lead to genomic instability with obvious significance for tumour progression. Understanding these controls may also provide new strategies for treating cancer. Quiescent cells do not have potentially activatable CDKs required for mitosis and so would be resistant to treatments which could activate these protein kinases. Malignant cells which are unable to become properly quiescent could respond to such treatments by a premature entry into mitosis which would be lethal, selectively killing malignant cells. These studies provide a useful example of how work with simple organisms such as yeast and starfish can illuminate fundamental problems in human cells which may ultimately be of practical importance for improving clinical practice.

Cdk2 and newly synthesized cdc2 promote initiation of S-phase in Xenopus egg extracts

Cdk2 and newly synthesized cdc2 promote initiation of S-phase in Xenopus egg extracts

Differential effects of topical retinoic acid application on keratin K1 and filaggrin expression in mouse epidermis

Abstract Retinoic acid (RA) modulates epidermal homeostasis and affects differentiation-associated proteins such as keratin K1 and filaggrin. Because results from in vitro and in vivo studies have been conflicting with respect to RA effects on keratinization, we examined the terminal differentiation of epidermal cell cohorts after RA stimulation in vivo. Pulse-labelling with 5-bromo-2-deoxyuridine (BrdU) was performed by intraperitoneal injection of mice immediately or at 16 h after a single topical application of 100 nmol RA. The cell cohort labelled at the time of RA application consisted of previously unperturbed cells exposed to RA after initiation of S-phase, whereas the cohort labelled 16 h after RA application consisted of cells stimulated into the S-phase by RA. Theses two cohorts of partially synchronized cells were followed for up to 72 h after BrdU labelling. Such labelling combined with keratin K1 or filaggrin expression was scored by paired immunofluorescence staining of skin sections. The onset of keratin K1 expression was unchanged in both series after RA treatment, while filaggrin appeared earlier than in controls. The differential effect of RA on the maturation markers was related to the proliferative activity, the increased cell turnover, and the shortened epidermal transit time. The onset of keratin expression appeared to be regulated before the postmitotic period, whereas filaggrin expression appeared to be regulated during the late phase of the maturation process, thus being influenced by the actual epidermal kinetics and structural alterations. These results suggested that the effect of RA on epidermal differentiation is secondary to its effect on proliferation, as determined by the altered cellular age distribution following regenerative proliferation.

Induction of v- mos and activated Ha- ras oncogene expression in quiescent NIH 3T3 cells causes intracellular alkalinisation and cell-cycle progression

We have tested the effect of the expression of the v- mos oncogene, and the activated human Ha- ras oncogene and its proto-oncogene form on the intracellular pH and on cell cycle progression. The Ha- ras proto-oncogene and the oncogenes under the transcriptional control of the MMTV-LTR promoter were introduced into NIH 3T3 cells. The cells were synchronized at g 0/g 1 in low-serum medium and the transfected genes were induced by glucocorticoid hormone addition to the growth medium. Expression of the v- mos and the activated form of Ha- ras oncogenes mimics the effect of growth factors and activates the Na +/H + antiporter. The oncogenes increase the internal pH and provoke initiation of S-phase. The proto-oncogene form of Ha- ras does not induce intracellular alkalinisation and has only a weak mitogenic effect. Our results suggest the oncogenic proteins p21 ras and p31 mos influence the mitogenic signal transduction pathways at a point prior to the activation of the Na + /H + antiporter.

Requirement for c-ras proteins during viral oncogene transformation.

Many retroviral oncogenes have been classified into one of several categories based on structure, enzymology and cellular localization1. These genes originated from host cells and are probably derived from genes normally involved in the control of cell proliferation2. The cellular counterparts of three oncogenes have been identified as a growth factor or growth factor receptor3–6; related oncogenes include receptor-like membrane proteins which often express tyrosine kinase activity. These growth factor-related oncogenes are structurally and biochemically distinct from the membrane-associated ras gene family, which bind and hydrolyse GTP7–9. Oncogenes localized primarily in the cytoplasm which probably have serine kinase activity, have also been identified10–12. Although the structure and biochemistry of many oncogenes have been extensively studied, relatively little is known about the functional relationships of oncogene proteins within the cell. An opportunity to study such interaction is provided by the identification of a monoclonal antibody that neutralizes cellular ras proteins when microinjected into cells13. It has been shown previously that the injected antibody inhibits the initiation of S-phase in NIH 3T3 cells14. In the present study we injected this monoclonal antibody into NIH 3T3 cells transformed by a variety of oncogenes. The results show that transformation by three growth factor receptor-like oncogenes depends on c-ras proteins, while transformation by two cytoplasmic oncogenes appears to be independent of c-ras protein.

An oscillator cell cycle model needs no first or second chance event

Theoretical data is presented to support the view that a limit cycle oscillator model of the cell cycle (as originally described) which accounts for random triggering of replication, can also accomodate the experimental results which have caused the supporters of the Transition Probability model to introduce the occurrence of yet a second chance event. In particular, it explains why the addition of a suboptimal amount of mitogen in a split dose alters the probability of triggering at each treatment but, generally, has little effect on the delay before the initiation of S-phase. It is also confirmed that, other than in some exceptional circumstances, lowering the probability of triggering in the oscillator model may have little effect on the minimum “intermitotic” time (as required by the evidence of the Transition Probability proponents) and, moreover, the oscillator concept provides reason why this should be so.

The effect of cell mass on the cell cycle timing and duration of S-phase in fission yeast

ABSTRACT Two isotopic methods for measuring DNA replication in the fission yeast Schizosaccharomyces pombe are described. The first is a method for measuring the total quantity of [3H]uracil incorporated into DNA after pulse labelling. The second is a means of detecting DNA replication in single cells by autoradiography. Both of these techniques have been used to investigate the timing and duration of S-phase in a series of mutant strains whose cell mass at division varies over a 3-fold range. The results support the hypothesis that in S. pombe there are 2 different controls over the timing of S-phase: an attainment of a critical cell mass and a dependency upon the completion of the previous mitosis coupled with a short minimum time in G1. Strains whose cell mass at birth is above this critical level initiate DNA replication almost immediately after septation, that is, very soon after the previous mitosis. Strains whose cell mass at birth is below the critical level do not initiate replication until the critical cell mass is attained. The duration of S-phase has been estimated from the proportion of cells whose nuclei are labelled after a pulse of given duration. S-phase is short in S. pombe, lasting only about 0·1of a cell cycle in wild type. Cell mass at S-phase does not have any consistent effect on this length. We have also investigated the degree of synchrony of S-phase initiation in daughter cells, and have found that, in a cell cycle 240 min long, their S-phases are initiated within 1-2 min of each other. This result indicates that between sisters variability in the duration of the G1 phase is small compared with variability in the total cell cycle time, and argues against the hypothesis that the rate of cell cycle traverse is determined by a random transition in G1.

The effect of cell mass on the cell cycle timing and duration of S-phase in fission yeast.

Two isotopic methods for measuring DNA replication in the fission yeast Schizosaccharomyces pombe are described. The first is a method for measuring the total quantity of [3H]uracil incorporated into DNA after pulse labelling. The second is a means of detecting DNA replication in single cells by autoradiography. Both of these techniques have been used to investigate the timing and duration of S-phase in a series of mutant strains whose cell mass at division varies over a 3-fold range. The results support the hypothesis that in S. pombe there are 2 different controls over the timing of S-phase: an attainment of a critical cell mass and a dependency upon the completion of the previous mitosis coupled with a short minimum time in G1. Strains whose cell mass at birth is above this critical level initiate DNA replication almost immediately after septation, that is, very soon after the previous mitosis. Strains whose cell mass at birth is below the critical level do not initiate replication until the critical cell mass is attained. The duration of S-phase has been estimated from the proportion of cells whose nuclei are labelled after a pulse of given duration. S-phase is short in S. pombe, lasting only about 0.1 of a cell cycle in wild type. Cell mass at S-phase does not have any consistent effect on this length. We have also investigated the degree of synchrony of S-phase initiation in daughter cells, and have found that, in a cell cycle 240 min long, their S-phases are initiated within 1--2 min of each other. This result indicates that between sisters variability in the duration of the G1 phase is small compared with variability in the total cell cycle time, and argues against the hypothesis that the rate of cell cycle traverse is determined by a random transition in G1.

A control acting over the initiation of dna replication in the yeast Schizosaccharomyces pombe

The control of cell division in the yeast Schizosaccharomyces pombe appears to be quite different to that of any other eukaryotic organisms for it is usually exerted not at the initiation of S-phase but at that of mitosis. However, it has been suggested that a control over the initiation of S-phase does also exist but that its action is redundant whilst the mitotic control is operating. This study has chosen conditions in which the latter appears to be largely absent in order to study the cryptic S-phase control. The timing of S-phase has been studied in cells grown at varying rates under nitrogen limitation in a chemostat. It is found that under these conditions the control of cell division resembles that of other eukaryotes. As the dilution rate of the chemostat is reduced, all increase in the generation time can be accounted for by a lengthened G1 period. In contrast, the length of S + G2 remains invariant. Thus, there must indeed be a control acting in G1 in S. pombe. An analysis of the size of cells at different growth rates shows that the initiation of S-phase is correlated with a particular cell size.

Antigen Recognition by Primed T Lymphocytes in Vitro

Abstract Antigen-induced T cell activation can be separated into early recognitive events and later proliferative events. After in vitro challenge of primed lymphocytes with antigen, enhanced transmembrane transport of α-aminoisobutyric acid (14C-AIB), a nonutilizable amino acid, can be demonstrated within 4 to 8 hr, and is at or near maximum by 8 to 16 hr of culture. The response is antigen specific and requires that the primed animal is a genetic responder to the immunizing antigen. Therefore, AIB uptake was utilized as a probe of antigen recognition and compared to tritiated thymidine incorporation, a measure of DNA synthesis. Colchicine, cytochalasin B, and Ca++ depletion have been reported to block DNA synthesis in activated lymphocytes and to interfere with macrophage-T cell interaction. We confirmed that these agents effectively abrogated antigen-induced DNA synthesis under conditions which, in contrast, had little effect on antigen-induced AIB uptake. We interpret these results to indicate at least a two-signal model for antigen-induced T cell proliferation. An initial signal is generated by the interaction of antigen with its membrane receptor. Lymphocyte activation commences but at some step before the initiation of S-phase, a second critical event, which can be blocked by colchicine, cytochalasin B, and Ca++ depletion, must occur. Since those agents block macrophage-T cell binding, the second signal may be the result of that cell-cell binding.

Transient inhibition of initiation of S-phase associated with dimethyl sulfoxide induction of murine erythroleukemia cells to erythroid differentiation.

The murine erythroleukemia cell (MELC) line in suspension culture can be induced to differentiate to erythroid cells by various compounds, including dimethyl sulfoxide (Me2SO). Analysis of the cell cycle, during differentiation induced by Me2SO, using thymidine incorporation, thymidine labeling index, and relative DNA content per cell as measured by flow microfluorometry, demonstrates a transient inhibition of entry of cells into S-phase of the cell cycle which is detected as early as 5 hr and is maximal about 20 hr after beginning of nonsynchronous cultures. Furthermore, in the presence of Me2SO there is restricted binding of the intercalating dye propidium iodide to chromatin from MELC in G1 phase of the cell cycle, as early as 10 hr of culture. This restricted binding of propidium iodide to chromatin is observed in MELC cultured with other inducing agents, such as butyric acid and dimethyl-acetamide, but is not detected with an Me2SO-resistant cell line cultured with Me2SO.

On the Action of Medicines, Old and New

<h3>Abstract</h3> Maintenance of genome integrity depends on controlling the availability of DNA replication initiation proteins, e.g., CDT1, a component of the pre-replication complexes that regulates chromatin licensing for replication. To understand the evolutionary history of CDT1 regulation, we have identified the mechanisms involved in CDT1 dynamics. During cell cycle, CDT1a starts to be loaded early after mitotic exit and maintains high levels until the G1/S transition. Soon after the S-phase onset, CDT1a is rapidly degraded in a proteasome-dependent manner. Plant cells use a specific SCF-mediated pathway that relies on the FBL17 F-box protein for CDT1a degradation, which is independent of CUL4a-containing complexes. A similar oscillatory pattern occurs in endoreplicating cells, where CDT1a is loaded just after finishing the S-phase. CDT1a is necessary to maintain genome stability, an ancient strategy although unique proteins and mechanisms have evolved in different eukaryotic lineages to ensure its degradation during S-phase. <h3>Impact statement</h3> The DNA replication protein CDT1a is crucial for genome integrity and is targeted for proteasome degradation just after S-phase initiation by FBL17 in proliferating and endoreplicating cells of Arabidopsis