The probability of G1 cells to enter into S increases with their size while S length decreases with cell enlargement in Allium cepa

The distribution of cell surface area projection (cell size) has been measured at birth and at initiation of DNA synthesis in steady-state populations of Allium cepa root meristems. The conditional probability, P(I/G₁), that initiation occurs given that the event of being in g₁ also occurs has been estimated from these data. p(I/G₁) was found to increase when cells became larger. The distribution of G₁ duration has been constructed from indicated cell size distributions. The absolute frequencies of G₁ times showed a maximum in the zone of cells with short G₁ periods; about 14% of cells appear to enter into S with G₁ - 1 h. These results suggest that the increase of p(I/G₁) was due to cell enlargement and not to cell aging. By comparing the cell size distribution at initiation of S and at the end of this period, a drastic reduction of cell size variability during DNA replication was observed and both curves were seen as rather similar in shape although they obviously had different modal points. These observations support that there is a negative correlation between the initiation size and the duration of genome duplication, and that cells which initiate DNA synthesis with the same size have a similar replication time. From this hypothesis, a plot of S duration versus cell size at initiation of this period was constructed by comparing the distributions of cell size at start and end of replication; this plot was also consistent with the existence of a negative correlation between cell initiation size and S length.     

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.     

The CRL4DTL E3 ligase induces degradation of the DNA replication initiation factor TICRR/TRESLIN specifically during S phase

A DNA replication program, which ensures that the genome is accurately and wholly replicated, is established during G1, before the onset of S phase. In G1, replication origins are licensed, and upon S phase entry, a subset of these will form active replisomes. Tight regulation of the number of active replisomes is crucial to prevent replication stress-induced DNA damage. TICRR/TRESLIN is essential for DNA replication initiation, and the level of TICRR and its phosphorylation determine the number of origins that initiate during S phase. However, the mechanisms regulating TICRR protein levels are unknown. Therefore, we set out to define the TICRR/TRESLIN protein dynamics throughout the cell cycle. Here, we show that TICRR levels are high during G1 and dramatically decrease as cells enter S phase and begin DNA replication. We show that degradation of TICRR occurs specifically during S phase and depends on ubiquitin ligases and proteasomal degradation. Using two targeted siRNA screens, we identify CRL4^DTL as a cullin complex necessary for TICRR degradation. We propose that this mechanism moderates the level of TICRR protein available for replication initiation, ensuring the proper number of active origins as cells progress through S phase.     

Critical nuclear DNA size and distribution associated with S phase initiation. Peripheral location of initiation and termination sites

Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology and autoradiography grain patterns between middle G1 and middle S phases. Our results show two distinct [3H]-thymidine labeling patterns. The first "peripheral" labeling pattern has a characteristic nuclear size distribution, in contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of very early S phase. The sizes of the second labeling pattern--homogeneous or inhomogeneous grain distribution throughout the nucleus--are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size (reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel experimentation with rat liver cells-synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography--confirms the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near the nuclear periphery.     

A new compound which reversibly arrests T lymphocyte cell cycle near the G1/S boundary

A novel cell cycle blocking agent profoundly suppressed the proliferation of mitogen-stimulated T lymphocytes. The carboxythiazole derivative arrested cells in the G1 phase of the cell cycle but did not inhibit the induction of cell surface receptors for either interleukin-2 or transferrin. The uncoupling of transferrin receptor expression from DNA synthesis indicated that a previously undefined restriction point in the cell cycle has been identified which occurs after transferrin receptor expression in late G1 and just prior to the initiation of DNA replication in S phase. T cells incubated in an inhibitory dose of the carboxythiazole derivative resumed cell cycle progression subsequent to its removal, indicating that the compound reversibly arrests cells at the late G1 restriction point. In contrast to other techniques which have been inefficient in achieving T cell synchronization, T cells released from the block mediated by the carboxythiazole compound progress through S phase with a considerable degree of synchrony.     

Determination of growth inhibitory action point of interferon gamma on WISH cells in cell cycle progression and the window of responsiveness of the cells to the interferon

We had earlier shown that human foetal epithelial cells (WISH), growth-inhibited by interferon gamma (IFNgamma), were reversibly detained at a point prior to DNA synthesis. In the present study, we determined the window of action of IFNgamma in the G1 phase duration and the exact point of detention of WISH cells in cell cycle progression with respect to the known points of detention by the inhibitors of DNA replication initiation (aphidicolin and carbonyl diphosphonate) and of activation of replication protein A (6-dimethylaminopurine), of which RPA activation being the earlier event compared to DNA replication initiation in cell cycle progression. WISH cells, which were released from IFNgamma-induced arrest, permeabilised and exposed independently to these inhibitors show that IFNgamma detains WISH cells prior to initiation of DNA synthesis. Further, exposure of IFNalpha-synchronized (at G0/G1) or mimosine-synchronized (at G1/S) WISH cells to IFNgamma, which was added at different time points post-release from the synchronizing agent, showed that the cells were promptly responsive to the growth inhibitory action of IFNgamma only during the first 11h in G1 phase. Taken together, these results suggest that IFNgamma inhibits growth of WISH cells by detaining them at a point prior to initiation of DNA synthesis and that the IFN acts within the first 11h in G1 phase of the cell cycle.     

Early and late replicating DNA involved in the G1 to S transition in Allium cepa L meristematic cells.

The involvement of portions of the genome replicated at different times of the S period in the regulation of the G1 to S transition was analyzed in Allium cepa L meristem cells. For this, DNA bromosubstitution confined to discrete portions of a previous S period followed by anoxic UVA irradiation (300-400 nm light) was performed in synchronous cells. Sequences replicated in late S appeared to be involved in the positive regulation of the initiation of replication. Hence, cells were prevented from initiating replication if irradiated at mid G1 only when the DNA sequences replicated in the last third of the previous S period were bromosubstituted. Cycloheximide-induced inhibition of protein synthesis at late G1 also prevented the G1 to S transition. Sequences replicated in mid S appeared unrelated to any control of the initiation of replication. On the other hand, sequences replicated in the first third of the S period seemed to be involved in the negative regulation of the initiation of replication, since irradiation after previous bromosubstitution of early replicating DNA sequences advanced G1 cells into the next S phase and increased the proliferative fraction of the population. Finally, the simultaneous inactivation of DNA sequences involved in both positive and negative regulation of replication allowed the cells to enter into S.     

Regulation of the initiation step of DNA replication by cyclin-dependent kinases

Cyclin-dependent kinases (CDKs) play a central role in the regulation of cell cycle progression in eukaryotes. The onset of S phase, the initiation of chromosomal DNA replication, is a major cell cycle event that is regulated by CDKs. Eukaryotic chromosomal DNA replication is highly regulated and occurs as a two-step reaction. The first reaction, known as licensing, is essential for DNA replication by making cell replication competent and occurs in G1 phase. Once cells enter S phase, licensed chromosomes initiate DNA replication through the action of two conserved protein kinases, S phase-specific CDK and Cdc7-Dbf4 (or Dbf4-dependent kinase). Our understanding of the regulatory mechanisms of DNA replication in model eukaryotes has advanced considerably in the past decade. In this review, we overview the regulation of DNA replication in the eukaryotic cell cycle, focusing specifically on how CDKs regulate the initiation step of DNA replication.     

Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex.

The transition from G1 to S phase of the cell cycle may be regulated by modification of proteins which are essential for initiating DNA replication. One of the first events during initiation is to unwind the origin DNA and this requires a single-stranded DNA binding protein. RPA, a highly conserved multi-subunit single-stranded DNA binding protein, was first identified as a cellular protein necessary for the initiation of SV40 DNA replication. The 32 kDa subunit of RPA has been shown to be phosphorylated at the start of S phase. Using SV40 replication as a model, we have reproduced in vitro the S phase-dependent phosphorylation of RPA-32 and show that it occurs specifically within the replication initiation complex. Phosphorylated RPA-32 is predominantly associated with DNA. Phosphorylation is not a pre-requisite for association with DNA, but occurs after RPA binds to single-stranded DNA formed at the origin during the initiation phase. The protein kinase(s) which phosphorylates RPA-32 is present at all stages of the cell cycle but RPA-32 does not bind to the SV40 origin or become phosphorylated in extracts from G1 cells. Therefore, the cell cycle-dependent phosphorylation of RPA-32 may be regulated by its binding to single-stranded origin DNA during replication initiation.     

Localization of human Mcm10 is spatially and temporally regulated during the S phase

Mcm10 (Dna43) is an essential protein for the initiation of DNA replication in Saccharomyces cerevisiae. Recently, we identified a human Mcm10 homolog and found that it is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner and that it binds chromatin exclusively during the S phase of the cell cycle. However, the precise roles that Mcm10 plays are still unknown. To study the localization dynamics of human Mcm10, we established HeLa cell lines expressing green fluorescent protein (GFP)-tagged Mcm10. From early to mid-S phase, GFP-Mcm10 appeared in discrete nuclear foci. In early S phase, several hundred foci appeared throughout the nucleus. In mid-S phase, the foci appeared at the nuclear periphery and nucleolar regions. In the late S and G phases, GFP-Mcm10 was localized to nucleoli. Although (2)the distributions of GFP-Mcm10 during the S phase resembled those of replication foci, GFP-Mcm10 foci did not colocalize with sites of DNA synthesis in most cases. Furthermore, the transition of GFP-Mcm10 distribution patterns preceded changes in replication foci patterns or proliferating cell nuclear antigen foci patterns by 30-60 min. These results suggest that human Mcm10 is temporarily recruited to the replication sites 30-60 min before they replicate and that it dissociates from chromatin after the activation of the prereplication complex.     

Initiation of DNA replication at a nuclear matrix-attached chromatin fraction

It is still unclear what nuclear components support initiation of DNA replication. To address this issue, we developed a cell-free replication system in which the nuclear matrix along with the residual matrix-attached chromatin was used as a substrate for DNA replication. We found out that initiation occurred at late G1 residual chromatin but not at early G1 chromatin and depended on cytosolic and nuclear factors present in S phase cells but not in G1 cells. Initiation of DNA replication occurred at discrete replication foci in a pattern typical for early S phase. To prove that the observed initiation takes place at legitimate DNA replication origins, the in vitro synthesized nascent DNA strands were isolated and analyzed. It was shown that they were enriched in sequences from the core origin region of the early firing, dihydrofolate reductase origin of replication ori-beta and not in distal to the origin sequences. A conclusion is drawn that initiation of DNA replication occurs at discrete sub-chromosomal structures attached to the nuclear matrix.     

DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity

Translesion DNA synthesis (TLS) employs low-fidelity DNA polymerases to bypass replication-blocking lesions, and being associated with chromosomal replication was presumed to occur in the S phase of the cell cycle. Using immunostaining with anti-replication protein A antibodies, we show that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persist into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase ζ and Rev1. Analysis of TLS using a high-resolution gapped-plasmid assay system in cell populations enriched by centrifugal elutriation for specific cell cycle phases showed that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions can lag behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. Such a mechanism may function to maintain efficient replication, which can progress despite the presence of DNA lesions, with TLS lagging behind and patching regions of discontinuity.     

Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin.

Both the inhibitory effect of aphidicolin on the replicative alpha-polymerase and the reversibility of its action in vivo (Pedrali-Noy & Spadari, 1979, Biochem. Biophys. Res. Commun. 88, 1194-2002) allow the synchronization of cells in culture. Aphidicolin prevents G1 cells from entering the DNA synthetic period, blocks cells in "S" phase, allows G2, M and G1 cells to continue the cell cycle and to accumulate at the G1/S border. Aphidicolin is a more useful reagent than hydroxyurea and thymidine because it does not affect cell viability or "S" phase duration and does not interfere with the synthesis of dNTPs or DNA polymerases. In fact cells exposed to the drug continue to synthesize all three DNA polymerases alpha, beta and gamma as well as all dNTPs which, when the block is removed, are present at levels optimal for DNA initiation and replication. The technique is simple and can be applied to cells growing in suspension or monolayers and allows one to harvest large quantities of synchronized cells.     

Induction of DNA replication in mammalian G1 nuclei by a heat-inactivible component from permeable S phase cells; an assay system

The in vitro initiation of DNA replication was studied in permeable mammalian cells by a newly developed procedure. Pairs of monolayer cultures, one synchronized in G1 and the other in S phase, were incubated in a sandwich with assay solution, containing Triton X-100 for permeabilization and [3H]TTP as a tracer. After 1.5 h DNA synthesis was shown to be induced in 36 to 81% of the G1 nuclei. The inducing capacity of the S phase cultures was diminished by at least 50% after a 10 min exposure to 60 degrees C prior to incubation. The suitability application of this in vitro system for testing components that might effect the initiation of DNA replication is shown in an assay with G1 cultures where the addition of up to 1 mM Ap4A led to an increase of DNA synthesizing cells from 4 to 15%.     

Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner

The synchronization effects of the plant amino acid mimosine on proliferating higher eukaryotic cells are still controversial. Here, I show that 0.5 mM mimosine can induce a cell cycle arrest of human somatic cells in late G1 phase, before establishment of active DNA replication forks. The DNA content of nuclei isolated from mimosine-treated cells was determined by flow cytometry. The presence or absence of DNA replication forks in these isolated nuclei was then detected by DNA replication run-on assays in vitro. Treatment of asynchronously proliferating HeLa or EJ30 cells for 24 h with 0.5 mM mimosine resulted in a population synchronized in late G1 phase. S phase entry was inhibited by 0.5 mM mimosine in cells released from a block in mitosis or from quiescence. When added to early S phase cells, 0.5 mM mimosine did not prevent S phase transit, but delayed progression through late stages of S phase after a lag of 4 h, eventually resulting in a G1 phase population by preventing entry into the subsequent S phase. In contrast, lower concentrations of mimosine (0.1-0.2 mM) failed to prevent S phase entry, resulting in cells containing active DNA replication foci. The G1 phase arrest by 0.5 mM mimosine was reversible upon mimosine withdrawal. This synchronization protocol using 0.5 mM mimosine can be exploited for studying the initiation of human DNA replication in vitro.     

Infection of cells with human cytomegalovirus during S phase results in a blockade to immediate-early gene expression that can be overcome by inhibition of the proteasome

Cells infected with human cytomegalovirus (HCMV) after commencing DNA replication do not initiate viral immediate-early (IE) gene expression and divide before arresting. To determine the nature of this blockade, we examined cells that were infected 24 h after release from G(0) using immunofluorescence, laser scanning cytometry, and fluorescence-activated cell sorting (FACS) analysis. Approximately 40 to 50% of the cells had 2N DNA content, became IE(+) in the first 12 h, and arrested. Most but not all of the cells with >2N DNA content did not express IE antigens until after mitosis. To define the small population of IE(+) cells that gradually accumulated within the S and G(2)/M compartments, cells were pulsed with bromodeoxyuridine (BrdU) just prior to S-phase infection and analyzed at 12 h postinfection for IE gene expression, BrdU positivity, and cell cycle position. Most of the BrdU(+) cells were IE(-) and had progressed into G(2)/M or back to G(1). The majority of the IE(+) cells in S and G(2)/M were BrdU(-). Only a few cells were IE(+) BrdU(+), and they resided in G(2)/M. Multipoint BrdU pulse-labeling revealed that, compared to cells actively synthesizing DNA at the beginning of the infection, a greater percentage of the cells that initiated DNA replication 4 h later could express IE antigens and proceed into S. Synchronization of the cells with aphidicolin also indicated that the blockade to the activation of IE gene expression was established in cells soon after initiation of DNA replication. It appears that a short-lived protein in S-phase cells may be required for IE gene expression, as it is partially restored by treatment with the proteasome inhibitor MG132.     

DNA replication during a serum-induced S phase in primate CV-1 cells

We have investigated components of DNA replication in a serum-induced S phase of primate CV-1 cells. Using DNA fiber autoradiography, we found a relative decrease in the frequency of initiation events in mid-S compared with early and late S phase. The other components of DNA replication measured by autoradiography—synchrony of initiation events, size of replication units, incidence of bidirectional replication, and the rate of replication fork movement—remained constant through S phase. When fork movement was measured by density gradient analysis of BUdR- and [³H]-thymidine-substituted DNA, it was also found to remain constant. These results show that most components of DNA replication are invariable through a serum-induced S phase. The changes in initiation frequency support the view that it may be critical in the regulation of ongoing replication.     

Critical nuclear DNA size and distribution associated with S phase initiation

Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology and autoradiography grain patterns between middle G₁ and middle S phases: Our results show two distinct [³H]-thymidine labeling patterns. The first “peripheral” labeling pattern has a characteristic nuclear size distribution, in contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of very early S phase. The sizes of the second labeling pattern—homogeneous or inhomogeneous grain distribution throughout the nucleus—are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size (reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel experimentation with rat liver cells—synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography—confirms the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near the nuclear periphery.