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.     

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.

     

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.     

Evidence for chromosomal replicons as units of sister chromatid exchanges

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 m with a peak value around 0.3 to 0.6 m regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments. A concentration of units around 0.4 to 1.0 m was found for L/S SCD segment size distributions regardless of CP treatment with an apparent bimodal profile. Our in vivo data support the existence of a subunit organization of chromosomal replication with a basic functional unit being 0.3 to 0.6 m in size. In addition, we have found that this chromosomal unit of replication or chromosomal replicon does not seem to be functionally perturbed by the mutagen CP. We also found that small SCD segments of 0.4 to 0.7 m in length were involved in the formation of an SCE, suggesting that both spontaneous and CP-induced SCEs occur between chromosomal replicons. These findings provide direct cytogenetic evidence to support a replicon cluster/chromosomal replicon model for SCE formation.     

DNA fork displacement rate measurements in heated Chinese hamster ovary cells

DNA fork displacement rates (FDR) were measured in Chinese hamster ovary (CHO) cells heated at either 43.5 degrees C or 45.5 degrees C for various times. The inhibition of fork movement rate by heat was both time and temperature dependent, i.e., 10-20 min at 43.5 degrees C or 5 min at 45.5 degrees C was required to decrease the FDR to 20-30% of the control rate of 1 micron/min. Following heating, the reduced FDR was found to be constant for at least 75 min. The observed effects of heat on reduced rates of DNA replicon initiation and chain elongation and the increase in DNA with single-stranded regions could be explained by the heat sensitivity of the FDR. Any of these alterations in the DNA replication process may lead to many opportunities for abnormal DNA and/or protein interactions to occur which ultimately may lead to the observed formation of chromosomal aberrations.     

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.     

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.     

Replicon sizes in mammalian cells as estimated by an x-ray plus bromodeoxyuridine photolysis method.

A new method is described for estimating replicon sizes in mammalian cells. Cultures were pulse labeled with [3H]thymidine ([3H]TdR) and bromodeoxyuridine (BrdUrd) for up to 1 h. The lengths of the resulting labeled regions of DNA, Lobs, were estimated by a technique wherein the change in molecular weight of nascent DNA strands, induced by 313 nm light, is measured by velocity sedimentation in alkaline sucrose gradients. If cells are exposed to 1,000 rads of X-rays immediately before pulse labeling, initiation of replicon operation is blocked, although chain elongation proceeds almost normally. Under these conditions Lobs continues to increase only until operating replicons have completed their replication. This value for Lobs then remains constant as long as the block to initiation remains and represents an estimate for the average size of replicons operating in the cells before X-irradiation. For human diploid fibroblasts and human HeLa cells this estimated average size is approximately 17 micron, whereas for Chinese hamster ovary cells, the average replicon size is about 42 micron.     

Mechanism of killing Chinese hamster ovary cells heated in G1: effects on DNA synthesis and blocking in G2

To determine where in the cell cycle Chinese hamster ovary cells die following heating in G1, a mild hyperthermia treatment, i.e., 10 or 11.5 min at 45.5 degrees C, resulting in 40-50% cell kill was used. After a 7-14-h delay in G1, the cells heated in G1 eventually entered S phase and replicated all their DNA. Both an autoradiographic analysis with tritiated thymidine and a bromodeoxyuridine-propidium iodide bivariate analysis by flow cytometry revealed that both clonogenic and nonclonogenic cells were delayed in progression through S phase for at least 4 h. Then they completed replication of all their DNA and entered G2. Alkaline sucrose gradient sedimentation analysis revealed that these heated cells could complete replicon elongation into cluster-sized molecules of 120-160 S which persisted for 2-12 h after heating. However, further replicon elongation into multicluster-sized molecules greater than 160 S required an additional 12 h in heated cells compared to the 4 h needed in unheated control cells. Our results when compared with the literature suggest that when G1 cells are heated to a survival level of about 50%, the nonclonogenic cells recover from a long delay in G1, traverse S at a reduced rate, and then die either in G2 or as multinucleated cells after an aberrant division.     

Uncoupling of SV40 tsA replicon activation from DNA chain elongation by temperature shifts and aphidicolin arrest.

To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 40 degrees, to complete ongoing replication and returned to 32 degrees, to activate new replicons in the presence of the DNA chain elongation inhibitor aphidicolin. Upon further incubation at 40 degrees without the drug, 3H-dT was incorporated into SV40 FI DNA, almost to the extent seen with cells recovered in the absence of the drug. To determine whether DNA synthesis would begin from the origin, following the temperature-shifts-aphidicolin regimen, chains subsequently pulse-labeled with (alpha-32p)dGTP in isolated nuclei were analyzed for size distribution and genomic location. These chains reached up to 300-400 nucleotides in size, unlike the control which featured comparable amounts of label in long chains and Okazaki pieces. The nascent DNA of the drug-treated system could be chased into longer chains, indicating that it was a replicative intermediate; and it hybridized preferentially to an origin proximal fragment of AtuI- restricted SV40 DNA, demonstrating partial replicon synchronization. The data prove that T-antigen activates the SV40 replicon independent of DNA chain elongation and suggest means to study the mechanism of DNA chain priming at the origin.     

Selective and synchronous activation of early-S-phase replicons of Ehrlich ascites cells.

Twelve-hour exposure of G1 Ehrlich ascites cells to controlled hypoxia (200 ppm of O2 at 1 bar) suppressed replicon initiation. Synchronous cycling, beginning with a normal S phase, was released by reoxygenation immediately. The addition of cycloheximide at reoxygenation largely resuppressed, after a short initial burst, succeeding replicon initiations. Alkaline sedimentation analysis of growing daughter strand DNA, DNA fiber autoradiography, and analysis of the newly formed DNA demonstrated that normal chain growth and DNA maturation (replicon termination) in the initially activated replicons continued in the presence of cycloheximide. After 2 to 3 h, a low level of cycloheximide-insensitive background replication emerged out of the then-ebbing single surge of activity of the initially released replicons.     

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.     

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.     

Inactivation of DNA replication origins by the cell cycle regulator, trigonelline, in root meristems of Lactuca sativa

 The effects of trigonelline (TRG) on the cell cycle in root meristems of Lactuca sativa L. were examined in the knowledge that TRG is a cell cycle regulator that causes cell arrest in G2, and prevents ligation of replicons in S-phase. The hypothesis was tested that continuous exposure to TRG would perturb DNA replication which, in turn, would lengthen the cell cycle and impair root elongation. Using DNA fibre autoradiography, mean replicon size was 31 and 13 μm in the TRG (3 mM) and control treatments, respectively. Trigonelline also resulted in a lengthening of both S-phase and the cell cycle and a decrease in primary root elongation. Hence, replicon inactivation was responsible for the protracted S-phase. Trigonelline treatment also resulted in a 1.6-fold increase in fork rate (13.8 μm h⁻¹) compared with the control (8.4 m h⁻¹). The faster fork rate in the larger replicons is in accord with the highly significant positive relationship already established between fork rate and replicon size for various unrelated higher plants. Received: 11 October 1999 / Accepted: 23 December 1999     

Nuclear matrix support of DNA replication

In higher eukaryotic cells, DNA is tandemly arranged into 10(4) replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the nuclear matrix at the bases of the loop. During late G1 phase, the replication origins are associated with the nuclear matrix and dissociated after initiation of replication in S phase. Clusters of several replicons are operated together by replication factories, assembled at the nuclear matrix. During replication, DNA of each replicon is spooled through these factories, and after completion of DNA synthesis of any cluster of replicons, the respective replication factories are dismantled and assembled at the next cluster to be replicated. Upon completion of replication of any replicon cluster, the resulting entangled loops of the newly synthesized DNA are resolved by topoisomerases present in the nuclear matrix at the sites of attachment of the loops. Thus, the nuclear matrix plays a dual role in the process of DNA replication: on one hand, it represents structural support for the replication machinery and on the other, provides key protein factors for initiation, elongation, and termination of the replication of eukaryotic DNA.     

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.     

Size maturation process of nascent DNA intermediates into chromosomal-sized DNA in Tetrahymena pyriformis macronuclear DNA replication

An analysis was made of the size maturation process of nascent DNA intermediates in macronuclear DNA replication of Tetrahymena pyriformis. The first discrete size class of nascent intermediates larger than Okazaki fragments were replicon-sized DNA (about 2 X 10(7) D single-stranded (ss) DNA) and accumulated in cells treated with cycloheximide. On removal of cycloheximide, the replicon-sized intermediates were converted to middle-sized intermediates (about 10 X 10(7) D ssDNA) and then merged into chromosomal-sized DNA. As indicated by either aphidicolin inhibition or the technique of the photolysis of bromodeoxyuridine (BrdU)-substituted DNA with long-wave ultraviolet light, four to eight replicon-sized intermediates were joined together to form a middle-sized intermediate after rapid sealing by DNA synthesis of the late-replicating regions located between adjacent replicon-sized intermediates. The late-replicating regions may represent the short gaps or terminal regions where DNA synthesis was retarded by cycloheximide, since the size of late-replicating regions was suggested to be shorter than the replicon size by DNA fiber autoradiography. Therefore, it is probable that four to eight completed replicons are joined as a group such as a replicon cluster, as has been reported in DNA replication of other eukaryotic cells.     

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.     

Initiating the Uninitiated: Replication of damaged DNA and Carcinogenesis

Cancer is a genetic disease and carcinogenesis is the process whereby the relevant genetic alterations are acquired. Environmental carcinogens may damage DNA to induce mutations and chromosomal aberrations as permanent heritable changes in the genome that initiate carcinogenesis. For many carcinogens initiation of carcinogenesis requires the initiation of DNA replication suggesting that genetic alterations are fixed in the genome during replication of damaged DNA. It is of great interest to understand the mechanisms whereby carcinogen-induced damage to DNA causes mutations and chromosomal aberrations, and how cells may resist such events. It is clear now that cells express a complex repertoire of responses to DNA damage including several pathways of DNA repair and cell cycle checkpoints that protect against carcinogenesis. This commentary is concerned with the protective influence of DNA damage checkpoints that delay or arrest progression through the cell division cycle and especially with the responses of S phase cells to the environmental carcinogens UV and benzo[a]pyrene diolepoxide I (BPDE). Recent studies indicate that checkpoint responses may act at the very point of replication of damaged DNA to slow DNA chain elongation, inhibit replicon initiation, and suppress initiation of carcinogenesis.