Enrichment and visualization of small replication units from cultured mammalian cells

DNA from cultured Chinese hamster cells has been fractionated to yield a population of DNA enriched for replicating molecules. Molecules containing replication structures were analyzed by electron microscopy, and replicon size was estimated. The enrichment procedure takes advantage of single-stranded regions characteristic of replicating molecules, and the greater affinity of mercuric ion for single-stranded rather than native DNA. After interaction with low concentrations of HgCl2, DNA with bound mercury is separated from the bulk of the DNA by virtue of its increased buoyant density in an isopycnic Cs2SO4 gradient. When DNA from cells labeled with [3H]thymidine for 45 s is interacted with HgCl2 and banded in Cs2SO4, the DNA with the highest specific activity is found in a dense region of the gradient. The high specific activity DNA behaves kinetically like nascent DNA since the radioactivity can be chased into main band if the cells are incubated for a further 2 h in excess unlabeled thymidine. Electron microscope analysis of the DNA in the enriched fraction confirmed that it contains a substantial fraction of molecules with replication structures. The level of enrichment is about 25-fold compared to unfractionated DNA or DNA taken from the main band of the Hg++/Cs2SO4 gradient. Of the replicating molecules visualized, 85% possessed a single replication structure. All molecules with multiple replication forms contained replicon sizes less than 5 micron, ranging from 0.2 to 4.5 micron. Replicon size was determined by measuring the distance from the center of one replication structure to the center of the adjacent replication structure on the same molecule. The replicons observed in this study are far smaller than can be detected by DNA fiber autoradiography and are in the same size range as the very small replication units reported in embryonic systems.     

Defective DNA synthesis in megaloblastic anaemia. Studies employing velocity sedimentation in alkaline sucrose gradients.

1. Autoradiographic experiments revealed that the average size of the replicating unit (replicon) in human phytohaemagglutunin-stimulated lymphocytes is 45 (+/- 1.3) micron. 2. A 5 min pulse of [3H]thymidine labelled DNA chains of approximately 40 S (15 micron) in control lymphocytes as revealed by velocity sedimentation in alkaline sucrose density gradients. Upon chasing in the absence of [3H]-thymidine the labelled DNA increased in size. By 6 h the bulk of the label co-sedimented with full-sized chromosomal DNA. 3. In untreated lymphocytes from patients with megaloblastic anaemia due to vitamin B-12 or folate deficiency or lymphocytes treated with methotrexate (10(-5) M) or hydroxyurea (5 . 10(-4) M) the increase in size of pulse-labelled DNA was slower than in control cells. 4. The block in maturation of pulse-labelled DNA to bulk DNA was not permanent. At 24 h of chase 75-80% of the pulse-label in both control and megaloblastic lymphocytes co-sedimented with bulk DNA. 5. We conclude that the lesions seen in DNA synthesis in megaloblastic anaemia due to folate or vitamin B-12 deficiencies occur through impaired biosynthesis of nucleotide precursors of DNA. Possible explanations of why the defects in DNA synthesis cause altered morphology of proliferating cells in megaloblastic anaemia are suggested.     

Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human Cells

In proliferating cells, DNA synthesis must be performed with extreme precision. We show that groups of replicons, labeled together as replicon clusters, form stable units of chromosome structure. HeLa cells were labeled with 5-bromodeoxyuridine (BrdU) at different times of S phase. At the onset of S phase, clusters of replicons were activated in each of ~750 replication sites. The majority of these replication “foci” were shown to be individual replicon clusters that remained together, as stable cohorts, throughout the following 15 cell cycles. In individual cells, the same replication foci were labeled with BrdU and 5-iododeoxyuridine at the beginning of different cell cycles. In DNA fibers, 95% of replicons in replicon clusters that were labeled at the beginning of one S phase were also labeled at the beginning of the next. This shows that a subset of origins are activated both reliably and efficiently in different cycles.

The majority of replication forks activated at the onset of S phase terminated 45–60 min later. During this interval, secondary replicon clusters became active. However, while the activation of early replicons is synchronized at the onset of S phase, different secondary clusters were activated at different times. Nevertheless, replication foci pulse labeled during any short interval of S phase were stable for many cell cycles. We propose that the coordinated replication of related groups of replicons, that form stable replicon clusters, contributes to the efficient activation and propagation of S phase in mammalian cells.

     

Application of hypoxia-induced shut down of replicon initiation to the analysis of replication intermediates in Ehrlich ascites cells

Cultured Ehrlich ascites cells were subjected to hypoxic conditions for about 2 h, then reaerated or allowed to remain hypoxic. The newly formed DNA of hypoxic or reaerated cells was labeled with [3H]thymidine using different pulse and pulse/pulse-chase protocols. The chain length distribution of the labeled DNA molecules was analysed by sedimentation after lysing the cells on the top of alkaline sucrose gradients. The results indicated that the hypoxia effectively and reversibly suppressed the initiation of new replication units. Initiation, growth and integration of Okazaki pieces into active replicons was not noticeably affected. In marked contrast to aerobic cells, the use of hypoxic cells allows the separation of Okazaki pieces as a distinct class of pulse labeled short DNA chains. Short daughter DNA of very recently initiated replicons did not interfere at pulse times shorter than 4 min. For examination of the newly initiated replicons it seems favourable to trigger a burst of initiations by reaeration.     

DNA replication in Physarum polycephalum: electron microscopic and autoradiographic analysis of replicating DNA from defined stages of the S-period.

Electron microscopic and autoradiographic analysis of replicating DNA from Physarum showed that replication occurs at a rate of 0.4 micron/min/per replicon and that replicons of size 10--15 mu occur in temporal clusters with an average of about 4 replicons per cluster. These results are compared with previous hydrodynamic measurements and with those obtained in other organisms.     

Initiation points for cellular deoxyribonucleic acid replication in human lymphoid cells converted by Epstein-Barr virus.

Replicon size was estimated in two Epstein-Barr virus (EBV)-negative human lymphoma lines, BJAB and Ramos, and four EBV-positive lines derived from the former ones by infection (conversion) with two viral strains, B95-8 and P3HR-1. Logarithmic cultures were pulse-labeled with [3H]thymidine, and the deoxyribonucleic acid was spread on microscopic slides and autoradiographed by the method of Huberman and Riggs. After developing, replication forks were visualized as silver grain tracks on the autoradiograms. Average replicon size was estimated by scoring the number of replication forks per constant length of deoxyribonucleic acid and by measuring distances between centers of adjacent tracks, followed by detailed statistical analyses. Three of the four EBV-converted cell lines, BJAB/B95-8, Ra/B95-8, and Ra/HRIK, were found to have significantly shorter replicons (41, 21, 54% shorter, respectively), i.e., more initiation points, than their EBV-negative parents. BJAB/HRIK had replicons which were only slightly shorter (11%) than those of BJAB. However, analysis of track length demonstrated that extensive track fusion occurred during the labeling of BJAB/ HRIK, implying that its true average replicon size is shorter than the observed value. The results indicate that in analogy to simian virus 40, EBV activates new initiation points for cellular DNA replication in EBV-transformed cells.     

Replication of chromosomal DNA in diploid Drosophila melanogaster cells cultured in vitro

Replication rate and replicon sizes in chromosomal DNA of in vitro cultured diploid D. melanogaster cells were determined using autoradiography of ³H-thymidine labeled DNA. Synthesis of DNA in euchromatic and heterochromatic regions of Drosophila diploid cells occurs at different periods of the S phase which lasts 10 h. During the first 4 h the synthesis is observed only in euchromatic regions. The heterochromatic synthesis starts shortly before the synthesis in euchromatic regions is completed and lasts for 6 h until the end of the S phase. The cells were synchronized by 5fluorodeoxyuridine which blocked the diploid cell DNA synthesis. Synthesis was found to start simultaneously in most euchromatic replicons. In the majority of the replicons the synthesis started at a single point and proceeded bidirectionally. The average rate of DNA synthesis per fork was 12.5 m/h (38 kb). The mean distance between the middle points of adjacent labeled regions was 70 m (210 kb). The size of most replicons ranged from 40 to 120 m. — These estimates do not apply to the heterochromatic portions of the D. melanogaster genome since the measurements have been carried out on DNA preparations obtained during the first 2 h of the S phase. — On the average, a replicon can consist of 7 chromomeres since the size of a replicon in diploid cell chromosomal DNA and DNA length of a polytene chromomere average 210 and 30 kb, respectively.     

The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment

Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [³H]TdR for 30 min, and chased in medium containing 1 × 10⁻⁶ M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9–12 × 10⁶ D single-stranded (ss) DNA. When the cells were chased in medium with 1 × 10⁻⁵ M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 × 10⁻⁴ M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability.     

DNA fiber replication during a morphogenetic switch in the shoot meristematic cells of a higher plant

Chromosomal DNA fiber replication was investigated in the shoot meristem of mustard plants during the morphogenetic transition from the leaf-forming (vegetative) to the flower-forming (evoked) condition. The replicon size, determined using the modal class, was 15 micron in the vegetative meristem and shifted to 7.5 micron in the evoked meristem. The average fork rate was 1.05 micron.h-1 in the vegetative meristem and only slightly increased to 1.55 micron.h-1 during the morphogenetic switch. Replicon activation was asynchronous but the pattern of activation of replicons was the same in both kinds of meristems. Thus the shortening of the S phase at the floral transition in mustard was essentially achieved by an increase of the number of replicon origins per unit length of DNA. These results are similar to those obtained in amphibian and Drosophila embryogenesis.     

Relationship between DNA chain growth termination and replicon sizes in γ-irradiated mouse cells

Unirradiated mouse leukemic L5178Y cells were pulse-labeled with [³H]thymidine continuously for up to 150 minutes. The resulting DNA fiber autoradiograms showed a continuously increasing fiber length up to 70 μm. However, in cells irradiated with 500 to 2000 rad of γ-rays, the resulting DNA fiber lengths leveled off at approximately 35 μm. Independent experiments to estimate replicon size by measuring the center-to-center distance in tandem tracks yielded a value of 48 μm. These results are consistent with a model proposing that the average replicon size is equal to √2 times the plateau value of the average length of labeled DNA measured in irradiated cells.     

Similar replicon properties of higher plant cells with different S periods and genome sizes

Root meristematic cells of nine unrelated diploid higher plants with genome sizes that differ 82-fold and with S periods that differ 4-fold have similar replicon sizes and single replication fork rates that average 22 μm and 8 μm/h respectively. The average replicon size of 22 μm is near the 18 μm obtained by extrapolation of measurements, taken from DNA fiber autoradiograms, to zero pulse time with [³H]thymidine. The data suggest that the duration of S is determined by the minimal number of replicon families that function sequentially during DNA replication.     

The effect of bleomycin on DNA synthesis in human cells: evidence for grouping of initiation of replicons in the S-period

The effect of bleomycin (Blm) on DNA synthesis has been studied in a synchronous culture of human embryonic lung cells. The data obtained suggest that in the Blm presence in a medium (20 micrograms/ml) DNA synthesis initiation in new replicons is suppressed. The Blm action at different S-phase intervals has been shown to inhibit DNA synthesis unequally. Four discrete time intervals have been singled out in the course of the 10-hr S-phase in which a grouped initiation of replicon portions can be supposed. Together with the data on DNA replication in large-size replicon units (50-500 microns), the obtained results account well for the uneven DNA synthesis in S-phase, manifested by 3 or 4 peaks of [3H]-thymidine incorporation in pulse-labelled cells.     

Mode of inhibition of DNA replication in neocarzinostatin-treated HeLa cells

The effect of antitumor antibiotic neocarzinostatin on DNA replication in HeLa cells was studied by pulse-labeling of DNA with [3H]thymidine and sedimentation analysis of the DNA with alkaline sucrose gradients. The drug, which produced DNA damage, primarily inhibited the replicon initiation in the cells at low doses (less than or equal to 0.1 microgram/ml), and at high doses (greater than or equal to 0.5 microgram/ml) inhibited the DNA chain elongation. An analysis of the number of single-strand breaks of parental DNA, induced by neocarzinostatin, indicated that inhibition of the initiation occurred with introduction of single-strand breaks of less than 1.5 . 10(4)/cell, while inhibition of the elongation occurred with introduction of single-strand breaks of more than 7.5 . 10(4)/cell. Assuming that the relative molecular mass of DNA/HeLa cell was about 10(13) Da, the target size of DNA for inhibition of replicon initiation was calculated to be about 10(9) Da, such being close to an average size of loop DNA in the cell and for inhibition of chain elongation, 1-2 . 10(8) Da which was of the same order of magnitude as the size of replicons. Recovery of inhibited DNA replication by neocarzinostatin occurred during post-incubation of the cells and seemed to correlate with the degree of rejoining of the single-strand breaks of parental DNA. Caffeine and theophylline enhanced the recovery of the inhibited replicon initiation, but did not aid in the repair of the breaks in parental DNA.     

Inhibition of mammalian cell DNA synthesis by ionizing radiation

A semi-log plot of the inhibitory effect of ionizing radiation on the rate of DNA synthesis in normal mammalian cells yields a two-component curve. The steep component, at low doses, has a D0 of about 5 Gy and is the result of blocks to initiation of DNA replicons. The shallow component, at high doses, has a D0 of greater than or equal to 100 Gy and is the result of blocks to DNA chain elongation. The target size for the inhibition of DNA replicon initiation is about 1000 kb, and the target size for inhibition of DNA chain elongation is about 50 kb. There is evidence that the target for both components is DNA alone. Therefore, the target size for inhibition of DNA chain elongation is consistent with the idea that an effective radiation-induced lesion in front of the DNA growing point somehow blocks its advance. The target size for inhibition of DNA replicon initiation is so large that it must include many replicons, which is consistent with the concept that a single lesion anywhere within a large group (cluster) of replicons is sufficient to block the initiation of replication of all replicons within that cluster. Studies with radiosensitive human cell mutants suggest that there is an intermediary factor whose normal function is necessary for radiation-induced lesions to cause the inhibition of replicon initiation in clusters and to block chain elongation; this factor is not related to poly(ADP-ribose) synthesis. Studies with radiosensitive Chinese hamster cell mutants suggest that double-strand breaks and their repair are important in regulating the duration of radiation-induced inhibition of replicon initiation but have little to do with effects on chain elongation. There is no simple correlation between inhibition of DNA synthesis and cell killing by ionizing radiation.     

Activation of replicon origins as a possible target for cytokinins in shoot meristems of Sinapis

Whilst the cytokinins are important promoters of plant cell division in vitro and in vivo, their mode of action remains unknown. Here we report the results of a study showing that a single application of a low dose of a cytokinin to the shoot apical meristem of Sinapis alba L. activates new replicon origins in chromosomal DNA, resulting in the halving of replicon size, and synchronizes the activation of replicon origins. These effects cause a 3.5-fold shortening of the duration of chromosomal DNA replication (S phase of the cell cycle). We hypothesize that one of the proteins involved in the initiation of DNA replication is a target for cytokinins.Abbreviations BA N⁶-benzyladenine - F fork rate - R size ofmost replicons - Rs time taken for replicon to replicate its allotedDNA - TdR [³H]thymidine - Ts duration of S phase C. Houssa is grateful to I.R.S.I.A. for the award of a research fellowship. This research was supported by the Belgian Government (Concerted Research Actions and FRFC).     

Characterization of DNA replication at a restrictive temperature in a mouse DNA temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha activity

tsFT20 cells derived from a mouse mammary carcinoma cell line FM3A have temperature-sensitive DNA polymerase alpha activity (Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1761-1765). DNA replication in tsFT20 cells at the restrictive temperature (39 degrees C) has been characterized in detail. DNA-synthesizing ability of these cells was measured by [3H] thymidine incorporation and autoradiography. The incorporation of [3H]thymidine decreased rapidly after temperature shift-up, and the incorporation was less than 20% of the initial level after 4 h at 39 degrees C. The rapid decrease correlated well with the decrease in the grain number in the individual nucleus but not with the number of cells with labeled nuclei. Alkaline sucrose gradient sedimentation analysis and DNA fiber autoradiography revealed that DNA chain elongation proceeded normally within a replicon in the temperature-sensitive cells incubated at the restrictive temperature and the DNA elongation rate did not change during the incubation at the restrictive temperature up to at least 6 h. On the other hand, the maturation of replicon-sized DNA to higher molecular weight DNA was retarded or inhibited in the temperature-sensitive cells at the restrictive temperature. The analysis of the center to center distance between replicons by DNA fiber autoradiography revealed that the frequency of replicon initiation decreased in tsFT20 cells at 39 degrees C.     

Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus

In mammalian cells, it has been shown that adjacent multiple DNA replicons, termed a replicon cluster or a replicon domain, are replicated coordinately in a defined temporal order during the DNA synthetic (S) phase. However, no intranuclear structure of this replicon domain has been revealed in the nucleus labelled with [3H]thymidine at the limited resolution level of autoradiography. By immunofluorescent staining with antibody against 5-bromodeoxyuridine (BrdU), we succeeded in detecting novel, intranuclear ring-like structures of replicating replicon domains that were organized temporarily during the S phase of mammalian cells with incorporated BrdU.     

Excess thymidine accelerates nascent replicon maturation without affecting the rate of DNA synthesis

The effects of different concentrations of exogenously supplied dThd on DNA replication were investigated in seedlings of Pisum sativum. Nascent DNA was labeled with either [³H]dThd or [³H]dAdo in the presence of 1·10^?6, 1·10^?5 or 1·10^?4 M unlabeled dThd. The rate of DNA synthesis was determined by measuring the kinetics of radioactivity incorporation into trichloroacetic acid-precipitable material and the size of the nascent molecules was investigated using alkaline sucrose gradients. The results obtained showed that high concentrations of exogenously supplied dThd accelerated the joining of completed nascent replicons without affecting the rate of DNA synthesis. This observation strengthens the hypothesis that the dTTP pool size is one of the factors controlling the timing of nascent replicon maturation.     

Rate of DNA replication and size of replicons in human diploid cells]

The rate of DNA replication and the distances between initiation sites (size of replicons) have been studied in human cultured fibroblasts. The modified Huberman and Riggs technique of DNA fiber autoradiography has been used: the pulse-labelled regions were analysed in DNA fibers preliminarily labelled along the whole length. This enabled us: a) to analyse the arrangement of replicons along the length of labelled DNA fibers with the lengths of 200-750 micron, reaching 2700 micron in some cases; b) to select only single DNA molecules for the analysis. This technique decreases the danger of a mistake when minor labelled regions belonging to different DNA molecules are referred to the same one. The rate of DNA replication varies from 0.2 to 1.2 micron/min, the average of 0.6 micron/min. This conforms with findings of other authors. The distances between initiation sites vary from 15 to 140 micron with the modal interval of 50-60 micron. This value is twice higher than those obtained by other authors. The possible reasons for such difference are discussed.