Initiation Of DNA Synthesis In Cells Research Articles

Model-based analysis of DNA replication profiles: predicting replication fork velocity and initiation rate by profiling free-cycling cells.

Eukaryotic cells initiate DNA synthesis by sequential firing of hundreds of origins. This ordered replication is described by replication profiles, which measure the DNA content within a cell population. Here, we show that replication dynamics can be deduced from replication profiles of free-cycling cells. While such profiles lack explicit temporal information, they are sensitive to fork velocity and initiation capacity through the passive replication pattern, namely the replication of origins by forks emanating elsewhere. We apply our model-based approach to a compendium of profiles that include most viable budding yeast mutants implicated in replication. Predicted changes in fork velocity or initiation capacity are verified by profiling synchronously replicating cells. Notably, most mutants implicated in late (or early) origin effects are explained by global modulation of fork velocity or initiation capacity. Our approach provides a rigorous framework for analyzing DNA replication profiles of free-cycling cells.

Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression.

Eukaryotic DNA synthesis initiates from multiple replication origins and progresses through bidirectional replication forks to ensure efficient duplication of the genome. Temporal control of initiation from origins and regulation of replication fork functions are important aspects for maintaining genome stability. Multiple kinase-signaling pathways are involved in these processes. The Dbf4-dependent Cdc7 kinase (DDK), cyclin-dependent kinase (CDK), and Mec1, the yeast Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related checkpoint regulator, all target the structurally disordered N-terminal serine/threonine-rich domain (NSD) of mini-chromosome maintenance subunit 4 (Mcm4), a subunit of the mini-chromosome maintenance (MCM) replicative helicase complex. Using whole-genome replication profile analysis and single-molecule DNA fiber analysis, we show that under replication stress the temporal pattern of origin activation and DNA replication fork progression are altered in cells with mutations within two separate segments of the Mcm4 NSD. The proximal segment of the NSD residing next to the DDK-docking domain mediates repression of late-origin firing by checkpoint signals because in its absence late origins become active despite an elevated DNA damage-checkpoint response. In contrast, the distal segment of the NSD at the N terminus plays no role in the temporal pattern of origin firing but has a strong influence on replication fork progression and on checkpoint signaling. Both fork progression and checkpoint response are regulated by the phosphorylation of the canonical CDK sites at the distal NSD. Together, our data suggest that the eukaryotic MCM helicase contains an intrinsic regulatory domain that integrates multiple signals to coordinate origin activation and replication fork progression under stress conditions.

The effects of soybean agglutinin binding on the corneal endothelium and the re-establishment of an intact monolayer following injury – A short review

This short review summarizes the localization and effects of the plant lectin soybean agglutinin (SBA) on the injured and non-injured organ-cultured rat corneal endothelium. Although the tissue exists as a non-cycling monolayer on the posterior corneal surface a circular freeze injury promotes wound repair as cells initiate DNA synthesis, mitosis and migration. As a result, by 24 h post-injury, endothelial cells express a surface protein that binds SBA in a diffuse punctate pattern, which by 48 h after injury, becomes confined to the cell periphery. As healing proceeds, SBA binding dramatically declines, such that, only scattered binding is observed by 72 h after wounding. In non-injured organ-cultured endothelia, weak SBA binding appears 24 h after explanation but becomes prominently detected around the cell periphery by 48 h. Incubating injured or non-injured endothelia in SBA leads to alterations in their cellular appearance due to the fact that lectin exposure results in the disruption of the actin cytoskeleton. Although this does not affect migration, treatment with either SBA or N-acetylgalactosamine (the SBA binding sugar) does interfere with the reestablishment of cell–cell contact. It is postulated that the surface protein that binds SBA is expressed during conditions that are stressful to the tissue. During organ-culture the protein's appearance suggests a cellular response to explantation in order to enhance or maintain monolayer integrity. In wound repair its appearance may serve to establish preliminary cell–cell contact during the restoration of the endothelial monolayer.

Genome-wide replication profiles of S-phase checkpoint mutants reveal fragile sites in yeast

The S-phase checkpoint kinases Mec1 and Rad53 in the budding yeast, Saccharomyces cerevisiae, are activated in response to replication stress that induces replication fork arrest. In the absence of a functional S-phase checkpoint, stalled replication forks collapse and give rise to chromosome breakage. In an attempt to better understand replication dynamics in S-phase checkpoint mutants, we developed a replication origin array for budding yeast that contains 424 of 432 previously identified potential origin regions. As expected, mec1-1 and rad53-1 mutants failed to inhibit late origin activation. Surprisingly however, 17 early-firing regions were not replicated efficiently in these mutants. This was not due to a lack of initiation, but rather to problems during elongation, as replication forks arrested in close proximity to these origins, resulting in the accumulation of small replication intermediates and eventual replication fork collapse. Importantly, these regions were not only prone to chromosome breakage in the presence of exogenous stress but also in its absence, similar to fragile sites in the human genome.

Heterogeneity in nuclear transport does not affect the timing of DNA synthesis in quiescent mammalian nuclei induced to replicate in Xenopus egg extracts.

Intact G0 nuclei from quiescent mammalian cells initiate DNA synthesis asynchronously in Xenopus egg extracts, despite exposure to the same concentration of replication factors. This indicates that individual nuclei differ in their ability to respond to the inducers of DNA replication. Since the induction of DNA synthesis requires the accumulation of replication factors by active nuclear transport, any variation in the rate of transport among nuclei could contribute to the variability of DNA replication. Using the naturally fluorescent protein allophycocyanin (APC) coupled with the nuclear localization sequence (NLS) of SV40 T antigen, as a marker of nuclear uptake, we show here that individual G0 nuclei differ in their rate of transport over a range of more than 20-fold. Surprisingly, this variation has no direct influence on the timing or extent of DNA synthesis. Similar results were obtained by monitoring the uptake of nucleoplasmin, a nuclear protein present at high levels in egg extracts. These experiments show that the initiation of DNA synthesis is not driven merely by the accumulation of replication factors to some threshold concentration. Instead, some other explanation is needed to account for the timing of initiation.

Regulation of mitogenesis by kinins in arterial smooth muscle cells

Recent evidence suggests that bradykinin (BK) plays a role in regulating neointimal formation after vascular injury. The present study examined the mechanism whereby BK regulates platelet-derived growth factor (PDGF) AB-induced mitogenesis in smooth muscle cells from rat mesenteric artery. BK, but not other activators of phosphoinositidase C (e.g., angiotensin II), inhibited PDGF-stimulated mitogenesis. The B1 receptor agonist des-Arg9-BK (DABK) was more potent than the B2 agonist BK; smaller BK fragments had no activity. In studies in which the B2 receptor antagonist HOE-140 {D-Arg0[Hyp3,beta-(2-thienyl)-Ala5,D-Tic7,Oic 8]BK} and the B1 receptor antagonist DHOE [[D-Arg0,Hyp3,beta-(2-thienyl)-Ala5,D-Tic7,Oi c8,des-Arg9]BK] were used, both receptors independently mediated inhibition of PDGF-induced mitogenesis. There was no evidence for metabolism of BK to DABK. The rank potency for activating phosphoinositidase C and increasing intracellular Ca2+ (BK > DABK) was opposite that for inhibiting mitogenesis (DABK > BK). Inhibition of cyclooxygenase did not prevent the kinin-mediated inhibition. Kinetic analysis of the cell cycle effects of kinins on PDGF-stimulated mitogenesis revealed that continuous exposure to DABK or BK was inhibitory even when added shortly before the cells initiated DNA synthesis (S phase). However, short-term exposure (5-60 min) to DABK or BK was inhibitory only when added after exposure to PDGF. These data suggest that the B1 and B2 receptors potently inhibited PDGF-stimulated mitogenesis and proliferation by activating an alternative signal transduction cascade not involving phosphoinositidase C or prostaglandins. The inhibition occurred at a point late in progression of the cell cycle from G1 to S and was dependent on the presence of kinins after exposure to PDGF.

Measurement of T-cell CD69 expression: a rapid and efficient means to assess mitogen- or antigen-induced proliferative capacity in normals.

We have analyzed the expression of the activation antigen CD69 on normal human T cells by flow cytometry following stimulation with mitogens and recall antigen. These data were compared to parallel studies assessing the proliferative response using the 3H-thymidine (3H-TdR) incorporation assay. Three different mitogens (PHA, ConA, and CD2/CD2R) induced maximal expression of CD69 at 24 h, which remained stable throughout the 72 h culture period. The mitogen-stimulated cells initiated DNA synthesis as determined by the 3H-TdR assay (72 h) while nonstimulated cells failed to upregulate CD69 or incorporate 3H-TdR. We next compared T cell CD69 expression (n = 12) following stimulation with either CD2/CD2R (5 micrograms/ml) or the recall antigen, tetanus toxoid (1:1500). Cell proliferation was determined by the 3H-TdR assay at 72 h (CD2/CD2R) or 120 h (tetanus toxoid). Evaluation of CD69 expression at 6 h predicted CD2/CD2R but not tetanus responder status as defined by 3H-TdR incorporation. However, when 4 known tetanus responders (3H-TdR) were evaluated over time, it was found that at 48 h the fluorescence intensity (of CD69) on tetanus-stimulated CD3+ cells increased markedly compared with nonstimulated cells (range of increase 43-850%). One individual whose cells failed to respond to tetanus toxoid (3H-TdR and CD69) did respond normally to CD2/CD2R. These observations suggest that flow cytometric evaluation of T cell CD69 expression following mitogen (6 h) or antigen (48 h) stimulation may provide an accurate screen of T-cell responsiveness in normals.

Ectopic E2F expression induces S phase and apoptosis in Drosophila imaginal discs.

Previous experiments suggest that a key event in the commitment of cultured mammalian cells to entering S phase is a rise in activity of the transcription factor E2F. In this report, we study the role of Drosophila E2F in imaginal disc cells in vivo, by examining the distribution of the endogenous protein and studying the consequences of ectopic E2F expression. First, we find that endogenous E217 falls from high to very low levels as cells initiate DNA synthesis during a developmentally regulated G1-S-transition in the eye disc. Second, we find that ectopic E2F expression drives many otherwise quiescent cells to enter S phase. Subsequently, cells throughout the discs express reaper (a regulator of apoptosis) and then die. Third, we find that ectopic E2F expression during S phase in normally cycling cells blocks their re-entry into S phase in the following cell cycle. Although we do not know the fate of these cells, we suspect that ultimately they are killed by ectopic E2F. Taken together, our results show that an elevation in the level of E2F is sufficient to induce imaginal disc cells to enter S phase. Furthermore, they suggest that the downregulation of E2F upon entry into S phase may be essential to prevent the induction of apoptosis.

IL-2-dependent induction of G1 cyclins in primary T cells is not blocked by rapamycin or cyclosporin A.

Autocrine stimulation of the IL-2 receptor (IL-2R) is required for commitment of a T cell to enter the cell cycle and may involve transmission of the IL-2R signal to cell cycle control proteins. Candidates for such proteins are the D-type cyclins which are expressed in G1. Short-term cultures of primary human T cells were used to show that expression of cyclins D2 and D3 is regulated by IL-2 in a concentration- and time-dependent manner. Cyclin D2 RNA was induced rapidly to peak levels well before initiation of DNA synthesis and gradually declined during the remainder of G1. Cyclin D3 RNA and protein showed a slower induction during G1 to maximal levels as cells initiated DNA synthesis that remained high throughout S phase. Induction of cyclins D2 and D3 was independent of the cyclosporin A-sensitive calcineurin pathway and of rapamycin-sensitive pathways, despite the ability of rapamycin to severely inhibit entry into S phase. These observations suggest that cyclins D2 and D3 may monitor the IL-2R signal but that their induction does not guarantee entry into S phase. Rapamycin was found to target a pathway late in G1 that is distal to induction of D-type cyclin gene expression but proximal to DNA replication, perhaps involving the function of the D-type cyclin proteins or their associated kinases.

Skin fibroblasts from aged fischer 344 rats undergo similar changes in replicative life span but not immortalization with caloric restriction of donors

We have compared the in vitro replicative life span and characteristics of immortalization of skin flbroblast cultures derived from ad libitum-fed and caloricrestricted Fischer 344 rats of 6, 24, and 29 months of age. Cells from all 6-, 24-, and 29-month-old animals showed a gradual decline in proliferative potential as evidenced by decreases in harvest density, in the fraction of cells initiating DNA synthesis, and in the number of population doublings per passage. These declines were accompanied by morphological changes including cell enlargement. The replicative life span prior to immortalization decreased significantly with donor age ( P < 0.0001), while caloric restriction had no effect on the cumulative population doubling level. Prior to immortalization mitotic cells from all cultures showed a normal rat karyotype. Postcrisis cultures tended to have more polyploid cells but there were no characteristic or specific chromosomal changes found in the cells with an immortalized phenotype. Interestingly, fibroblasts derived from caloric-restricted animals had a significantly slower growth rate through the tenth week after immortalization ( P < 0.0057). When these cultures were seeded at one-quarter the normal seeding density, to favor the outgrowth of the fastest growing cells, a population with a more “transformed” phenotype emerged.

Fundamentals of Cell Proliferation: Control of the Cell Cycle

Cell proliferation in higher eukaryotes is controlled by the extracellular environment and the state of differentiation. Many cells exist in a nondividing growth state termed quiescence. Some quiescent cells cannot proliferate and are said to be terminally differentiated. Others can be stimulated to divide in response to environmental signals or when cell replacement is needed. Finally, some cells undergo continual proliferation and differentiation.Growth regulatory factors generally act at specific stages of the cell cycle, most commonly during the first gap phase of the cell cycle. Once cells initiate DNA synthesis, they are generally committed to complete DNA replication. After DNA synthesis, additional signals determine whether cells in the last gap phase proceed through mitosis.In recent years, genes that appear to be critical for progression through the first two gap phases have been identified. Many are proto-oncogenes and therefore can neoplastically transform certain cells when mutated or inappropriately expressed. Growth factors that stimulate proliferation induce the expression of several proto-oncogenes; growth inhibitory factors often suppress proto-oncogene expression.As cells differentiate, the response to extracellular factors changes. In many cases, this may be due to intracellular controls that alter the response of certain proto-oncogenes to external signals.

Growth-related Gene Expression in Type 2 Alveolar Epithelial Cells

Our laboratory is studying mechanisms of growth control in alveolar type 2 cells. This highly differentiated cell is induced to proliferate in lungs of animals of all ages during various forms of growth and during the repair process after lung injury. Using type 2 (T2) cells isolated from adult and neonatal rat lungs and an SV40-T transfected T2 cell line, we have shown tha growth-arrested T2 cells constitutively express genes associated with G1 and S phase of the cell cycle, yet they do not efficiently translate the proteins encoded by these genes. This block of growth-related gene expression is post-transcriptional and appears to involve mechanisms that control translation, perhaps at the level of initiation. Furthermore, growth-arrested T2 cells initiate DNA synthesis; however, the cells do not complete the cell cycle, suggesting that they are arrested in a late stage, perhaps the G1/S border. Differential screening of a cDNA library of growth-arrested T2 cells with DNA from growing and growth-arrested T2 cells has identified four families of genes preferentially expressed in the growth-arrested cells. These genes, which are in the process of being characterized, may be responsible for the unusual type of growth arrest demonstrated by T2 cells.

Taxol inhibits stimulation of cell DNA synthesis by human cytomegalovirus

The microtubule (MT)-stabilizing drug, taxol, inhibited human cytomegalovirus (CMV)-initiated cell DNA synthesis by up to 100% in serum-arrested mouse embryo (ME) fibroblasts that were abortively infected by CMV. Taxol concentrations known to increase MT polymerization and to stabilize existing MTs (10 to 20 μg/ml) blocked CMV-stimulated cell DNA synthesis, while taxol concentrations of 2.5 μg/ml, or less, did not. Taxol maximally inhibited CMV initiation of cell DNA synthesis when added 3 h after virus infection and inhibited this initiation by greater than 50% when added up to 12 h after CMV infection. Control experiments suggest that taxol specifically inhibited CMV-stimulated cell DNA synthesis. Pretreatment of CMV stock with taxol did not reduce the stimulatory effect of CMV on cell DNA synthesis and taxol had no detectable effect on CMV-specific early protein synthesis. Moreover, taxol did not appear to alter thymidine pool sizes, affect cell viability, or compromise the DNA synthetic machinery in CMV-infected cells. Since taxol increases tubulin polymerization and inhibits MT disassembly, these results suggest that dynamic changes in MTs or in the pool of free tubulin subunits are necessary for CMV to stimulate cell entry into a proliferative cycle.

Calcium ion-dependent proliferation of L1210 cells in culture

Maximum growth of L1210 cells in culture required the presence of free extracellular calcium ions. Reducing the free extracellular calcium ion concentration with EGTA served to decrease the growth rate of the cells. The decrease in cell growth was not due to cell death but rather due to the “pile-up” of the L1210 cells in the GO/G1 phase of the cell cycle. With the readdition of excess calcium ions, there was a lag period of 3 to 6 hours before the L1210 cells initiated DNA synthesis or transited from the GO/G1 phase to S-phase. Cells enriched for S and G2/M phases by elutriation and which were incubated in EGTA-containing culture medium, continued through the cell cycle and were blocked in GO/G1. These data indicate that the proliferation of L1210 cells in culture requires a calcium ion-dependent process to allow movement from the GO/G1 to S-phase of the cell cycle.

Late G1 amino acid restriction point in human dermal fibroblasts.

Human dermal fibroblasts arrested in G0 by maintenance in medium supplemented with 0.1% serum were not restimulated to divide when fresh medium containing 10% dialyzed serum but lacking group B amino acids (cystine, isoleucine, lysine, phenylalanine and tyrosine) was added. Unlike rodent cells, the addition of fresh serum-supplemented medium lacking only isoleucine did not cause a growth arrest. The amino acid sensitive growth arrest in human fibroblasts was dependent both on presynchronization in G0 as well as a prestarvation for amino acids prior to stimulation with high serum. When cells were restimulated in the absence of amino acids, they arrested predominantly in G1, although a small percentage of cells entered early S phase. When medium containing a complete complement of amino acids was then added, cells initiated DNA synthesis following a minimum lag of 2-3 hr. Growth arrested cells initiated DNA synthesis even when complete unsupplemented medium was added, although the addition of high concentrations of insulin or 10% serum increased the rate of entry.

Use of low temperature for growth arrest and synchronization of human diploid fibroblasts

The growth kinetics of human diploid fibroblasts at two different temperatures were followed. Proliferation of exponentially growing cells is reduced and eventually stops upon incubation at low temperature (i.e. 30°C). The cells which are in S phase at the time of switching to low temperature complete their DNA synthesis and become arrested in the G 1 phase of the cell cycle. The arrested cells can be stimulated to proliferate by restoration of the optimal growth temperature (37°C). The kinetics of entry into S phase were investigated by measuring [ 3H]thymidine incorporation into TCA-precipitable material, by autoradiography and by flow cytofluorimetry. The synchronized cells initiate DNA synthesis at approximately 8 h and DNA synthesis peaks at 20.4±0.7 h after stimulation. In addition, the rates of UV-induced excision repair at 30°C and 37°C were compared. The results indicate that at 30°C the excision-repair process is operative but at a slightly reduced rate in comparison with repair at 37°C. This method will be useful for the study of S-phase-dependent processes, as well as for repair studies in the absence of cell division.

Permissive effect of cytochalasin B on DNA synthesis in concanavalin-A-treated lymphocytes.

Unfractionated or T-cell-enriched human lymphocytes can be stimulated to undergo DNA synthesis and mitosis by the addition of polyclonal cell activators such as the plant lectins phytohaemagglutinin and concanavalin A (ConA). Under conventional culture conditions stimulated cells cease proliferating only a few days after the first cells have initiated DNA synthesis. Cytochalasin B (CB), which is non-mitogenic per se, causes a prolongation of the period during which ConA stimulates DNA synthesis from normally 3-5 days to more than 3 weeks. The CB-induced prolongation of cell proliferation is clearly stage-specific in the sense that the CB effects are exerted after an initial period of 24 h and do not come into effect until 48 h after onset of ConA stimulation. In contrast, CB exerts a slight suppressive action on DNA synthesis between 24 h (when activated cells initiate DNA synthesis) and 48 h after onset of stimulation. These two separate effects of CB, i.e. augmentation of lymphocyte stimulation 48 h after stimulation, and suppression of stimulation before this point of time, are relatively independent of the concentration of CB.

Permissive effect of substratum contact on DNA synthesis in concanavalin A-treated lymphocytes.

The effects of contact with a solid surface (in the form of adhesive microcarrier beads (Cytodex)) on the induction of proliferation of human blood lymphocytes after exposure to concanavalin A (Con A) were investigated. The duration of the lag phase preceding DNA synthesis was identical in the presence and absence of beads. The kinetic course of initiation of DNA synthesis was exponential but with different rate constants both in the presence and in the absence of beads during the first 4 days of the culture period. Cytodex exerted its effect by increasing the proportion of cells initiating DNA synthesis in response to Con A. To elucidate whether lymphocytes responded to Con A exposure in a dose-dependent manner, cells were seeded at different concentrations and exposed to Con A for different times. At 36 and 48 h after start of stimulation the proportion of cells synthesizing DNA increased with increasing cell number in the cultures. In contrast, the portion of cells synthesizing DNA was markedly decreased at high cell densities 60, 72 and 96 h after start of stimulation. In the presence of microcarrier beads, however, the proportion of cells synthesizing DNA was proportional to the cell number in the cultures also after 60, 72 and 96 h.

Studies on cell division in mammalian cells: VI. A temperature-sensitive mutant blocked in both G1 and G2 phases of the cell cycle.

A temperature-sensitive mammalian cell cycle mutant with blocks in G1 and G2 phases of the cell cycle has been isolated in culture. When shifted from the permissive temperature of 33 degrees C to the nonpermissive temperature of 39 degrees C, the fraction of cells initiating DNA synthesis as well as the fraction of cells entering mitosis decreased rapidly. Combined cytophotometric and autoradiographic analysis on the cells at 39 degrees C showed that G1 cells, with the exception of those in late G1, were arrested in that phase. Cells is S phase at the time of temperature shift, together with the late g1 cells which subsequently entered S, continued through S into G2, but were blocked in that phase of the cell cycle and unable to initiate mitosis. Those cells already in mitosis completed cell division at 39 degrees C. The G1 block point of ts-550C was found to be located after the serum starvation and isoleucine deprivation arrest points, approximately 3 h before initiation of DNA synthesis.

Inhibition by 12-O-tetradecanoylphorbol-13-acetate of 17 beta-estradiol-induced initiation of DNA synthesis in rabbit endometrial cells in culture.

The initial effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 17 beta-estradiol on initiation of DNA synthesis by primary cultures of estrogen-responsive rabbit uterine epithelial cells were determined by autoradiography after a [3H]thymidine pulse. In contrast to the stimulatory response produced by estradiol, TPA (1.6 x 10(-7) M) reduced the fraction of cells synthesizing DNA for up to 48 h. Cells synthesizing DNA at the time TPA was added were found to be less sensitive to its inhibitory effect, suggesting that TPA blocked the cells from entering the S phase of the cell cycle. The cells were unresponsive to estradiol during the TPA-induced inhibitory period, although TPA did not interfere with the binding of [3H]estradiol to the hormonal whole-cell receptors. The results indicted that TPA acted to reduce the fraction of cells initiating DNa synthesis by a mechanism that appears to be independent of estrogen involvement.