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.     

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.     

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.     

Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci

According to the current paradigm, replication foci are discrete sites in the interphase nucleus where assemblies of DNA replication enzymes simultaneously elongate the replication forks of 10–100 adjacent replicons (each ∼100 kbp). Here we review new results and provide alternative interpretations for old results to show that the current paradigm is in need of further development. In particular, many replicons are larger than previously thought – so large that their complete replication takes much longer (several hours) than the measured average time to complete replication at individual foci (45–60 min). In addition to this large heterogeneity in replicon size, it is now apparent that there is also a corresponding heterogeneity in the size and intensity of individual replication foci. An important property of all replication foci is that they are stable structures that persist, with constant dimensions, during all cell cycle stages including mitosis, and therefore likely represent a fundamental unit of chromatin organization. With this in mind, we present a modified model of replication foci in which many of the foci are composed of clusters of small replicons as previously proposed, but the size and number of replicons per focus is extremely heterogeneous, and a significant proportion of foci are composed of single large replicons. We further speculate that very large replicons may extend over two or more individual foci and that this organization may be important in regulating the replication of such large replicons as the cell proceeds through S-phase. Received: 16 August 1999 / Accepted: 17 August 1999     

The effects of incorporated tritium and bromodeoxyuridine on the frequency of sister chromatid exchanges

Cells in third mitosis treated during the first cell cycle with ³H-TdR and during the next two cycles with BrdU (without ³H-TdR) show a typical pattern of chromosome differentiation which allows identification of sister chromatid exchanges occurring during the first (SCE₁, second (SCE₂) and third cycles (SCE₃). Chromosomes labeled only with ³H-TdR had the most SCEs; those labeled only with BrdU, the second highest number; and those labeled with ³H-TdR plus BrdU, the fewest. Since BrdU and ³H-TdR are well known inducers of SCEs, the relatively low frequency of exchanges produced by the combined action of these two compounds is paradoxical. — It is assumed that SCEs are generated by the abnormal recombination of double-strand DNA breaks occurring at the junctions between completely and partially duplicated replicon clusters. Thus, agents that induce absolute blocks to DNA fork displacement will favor the appearance of SCEs because double-strand breaks have more time to occur at junctions. Conversely, agents that inhibit the initiation of replication will decrease the probability of SCEs. Ionizing radiation delays the onset of cluster replication. Therefore, in ³H-TdR plus BrdU-substituted chromosomes the radiation from tritium may inhibit the appearance of BrdU-induced SCEs. Since the inhibition does not exist in chromosomes substituted only with BrdU, the frequency of SCEs in these elements is higher than in double-substituted chromosomes. During the first cell cycle the onset of cluster replication is normal. However, the incorporation of ³H-TdR in the replication fork may enhance the appearance of double-strand breaks, thus inducing a high frequency of SCEs.     

Studies of mammalian chromosome replication

Sister chromatids of metaphase chromosomes can be differentially stained if the cells have replicated their DNA semiconservatively for two cell cycles in a medium containing 5-bromodeoxyuridine (BrdU). When prematurely condensed chromosomes (PCC) are induced in cells during the second S phase after BrdU is added to the medium, the replicated chromosome segments show sister chromatid differential (SCD) staining. Employing this PCC-SCD system on synchronous and asynchronous Chinese hamster ovary (CHO) cells, we have demonstrated that the replication patterns of the CHO cells can be categorized into G₁/S, early, early-mid, mid-late, and late S phase patterns according to the amount of replicated chromosomes. During the first 4 h of the S phase, the replication patterns show SCD staining in chains of small chromosome segments. The amount of replicated chromosomes increase during the mid-late and late S categories (last 4 h). Significantly, small SCD segments are also present during these late intervals of the S phase. Measurements of these replicated segments indicate the presence of characteristic chromosome fragment sizes between 0.2 to 1.2 m in all S phase cells except those at G₁/S which contain no SCD fragments. These small segments are operationally defined as chromosome replicating units or chromosomal replicons. They are interpreted to be composed of clusters of molecular DNA replicons. The larger SCD segments in the late S cells may arise by the joining of adjacent chromosomal replicons. Further application of this PCC-SCD method to study the chromosome replication process of two other rodents, Peromyscus eremicus and Microtus agrestis, with peculiar chromosomal locations of heterochromatin has demonstrated an ordered sequence of chromosome replication. The euchromatin and heterochromatin of the two species undergo two separate sequences of decondensation, replication, and condensation during the early-mid and mid-late intervals respectively of the S phase. Similar-sized chromosomal replicons are present in both types of chromatin. These data suggest that mammalian chromosomes are replicated in groups of replicating units, or chromosomal replicons, along their lengths. The organization and structure of these chromosomal replicons with respect to those of the interphase nucleus and metaphase chromosomes are discussed.     

High resolution analysis of replication foci by conventional fluorescent microscopy. I. A study of complexity and DNA content of the foci

Newly replicated DNA segments (RDS) have been shown to form discrete foci in the mammalian nucleus. Comparison of the number of such foci in formaldehyde-fixed cell nucleus with estimated number of simultaneously active replication forks (RF) suggests that each replication focus contains a cluster of about 10 to 20 closely associated RF. That implied the cluster of synchronously activated replicons as the primary unit of mammalian DNA replication. It still remains unclear whether such clustering of RF does mean adjacency of the replicons in a genomic location (structural clustering, model 1), or it arises from transient clustering of the replicons from different DNA domains at the functioning replication machinery (functional clustering, model 2). In this study we used conventional fluorescence microscopy of the hypotonically treated nuclei preparations to investigate replication foci at the optical resolution limit. Human K562 cells were labeled with 5'-iododeoxyuridine for different time periods. We synchronized the cell culture with hydroxyurea to be able to measure an average increase in DNA content during labeling period using DNA cytometry. Under these conditions, RDS appear as multiple small foci (mini-foci, MF). Further studies revealed that most of such mini-foci of replication represent optical diffraction spots, which are standard in size and different in brightness. The number of the "spots" and variation of their brightness mostly depend on the extent of hypotonic treatment. Flow cytometry control of the synchronized cells peak movement allowed us to measure mean DNA content of the MF. In case of most effective hypotonic treatment, a MF contains about 40 Kbp of labeled DNA, and the general number of the MF approaches the number of replicons that are simultaneously active in a given moment of S-phase. Influence of the effect of hypotonic treatment on overall number of observed MF suggests that replication foci in early and mid S-phase cells do not represent stable structures, but rather arise from functional clustering of comparatively distant replicating regions, thus supporting model 2.     

Recovery from effects of heat on DNA synthesis in Chinese hamster ovary cells

The hyperthermic inhibition of cellular DNA synthesis, i.e., reduction in replicon initiation and delay in DNA chain elongation, was previously postulated to be involved in the induction of chromosomal aberrations believed to be largely responsible for killing S-phase cells. Utilizing asynchronous Chinese hamster ovary cells heated for 15 min at 45.5 degrees C, an increase in single-stranded regions in replicating DNA (as measured by BND-cellulose chromatography) persisted in heated cells for as long as replicon initiation was affected. Alkaline sucrose gradient analyses of cells pulse-labeled immediately after heating with [3H]thymidine and subsequently chased at 37 degrees C revealed that these S-phase cells can eventually complete elongation of the replicons in operation at the time of heating, but required about six times as long relative to control cells which completed replicon elongation within 4 h. DNA chain elongation into multicluster-sized molecules was prevented for up to 18 h in these heated cells, resulting in a buildup of cluster-sized molecules (approximately 120-160 S) mainly because of the long-term heat damage to the replicon initiation process. Utilizing bromodeoxyuridine (BrdU)-propidium iodide bivariate analysis on a flow cytometer to measure cell progression, control cells pulsed with BrdU and chased in unlabeled medium progressed through S and G2M with cell division starting after 2 h of chase time. In contrast, the majority of the heated S-phase cells progressed slowly and remained blocked in S phase for about 18 h before cell division was observed after 24 h postheat. Our findings suggest that possible sites for where the chromosomal aberrations may be occurring in heated S-phase cells are either (1) at the persistent single-stranded DNA regions or (2) at the regions between clusters of replicons, because this long-term heat damage to the DNA replication process might lead to many opportunities for abnormal DNA and/or protein exchanges to occur at these two sites.     

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.     

Analysis of chromosome replication by a BrdU antibody technique

Chromosome replication was studied without synchronization in human lymphocyte and amniotic cell cultures visualizing very short 5-bromodeoxyuridine (BrdU) pulses by an immunologic technique (BAT). The findings agree in general with those facts known from earlier BrdU staining techniques. The very high sensitivity of BAT was shown to allow the detection of replication in a band where 1 in 200 nucleotides is replaced by BrdU. The main observations are: though the replication patterns after BAT appear strange the bands correspond to those described by the Paris Conference (1971). At the beginning of the S-phase a stepwise onset of replication in only a subset of R-bands is confirmed. There is a considerable difference in the sensitivity between early and late S (S_E and S_L) for the detection of BrdU pulses. This difference probably reflects a different spatial arrangement of chromatin in R-bands as compared with G-bands below the level of cytogenetic analysis. The use of short pulses did not reveal any additional subdivision of S_E or S_L. The correspondence between chromosomal bands and replicon clusters is discussed briefly with respect to the different time they need for replication.     

Characterization of the Pre-meiotic S Phase through Incorporation of BrdU during Spermatogenesis in the Rat

Seminiferous tubules in mammals have histological arrangements defined by the associations between somatic cells and germ cells. The processes of DNA synthesis in meiotic and mitotic cells have different features that are not easily distinguishable through morphological means. In order to characterize the pre-meiotic S phase, 5-bromo-2’-deoxyuridine (BrdU) was injected intraperitoneally into Wistar rats, which were sacrificed 30 min, 2 hr, and 24 hr after injection. We found three different labeling patterns. One of these patterns was characterized by a distribution of the label in the form of speckles, most of which were associated with the nuclear envelope (labeling type I). We suggest that this pattern is due to mitotic DNA synthesis of type B spermatogonia. Labeling type II consisted of labeled foci scattered throughout the nuclear volume, which can be correlated with preleptotenic cells in pre-meiotic DNA synthesis. After 24 hr of incorporation, a third type of labeling, characterized by large speckles, was found to be related to cells in the “bouquet” stage; that is, cells in transition between the leptotene and zygotene phases. Our results indicate that BrdU incorporation induces different labeling patterns in the mitotic and pre-meiotic S phases and thus makes it possible to identify somatic and germinal cells.     

The Replication Domain Model: Regulating Replicon Firing in the Context of Large-Scale Chromosome Architecture

The “Replicon Theory” of Jacob, Brenner, and Cuzin has reliably served as the paradigm for regulating the sites where individual replicons initiate replication. Concurrent with the replicon model was Taylor's demonstration that plant and animal chromosomes replicate segmentally in a defined temporal sequence, via cytologically defined units too large to be accounted for by a single replicon. Instead, there seemed to be a program to choreograph when chromosome units replicate during S phase, executed by inititation at clusters of individual replicons within each segment. Here, we summarize recent molecular evidence for the existence of such units, now known as “replication domains”, and discuss how the organization of large chromosomes into structural units has added additional layers of regulation to the original replicon model.     

S Phase Progression in Human Cells Is Dictated by the Genetic Continuity of DNA Foci

DNA synthesis must be performed with extreme precision to maintain genomic integrity. In mammalian cells, different genomic regions are replicated at defined times, perhaps to preserve epigenetic information and cell differentiation status. However, the molecular principles that define this S phase program are unknown. By analyzing replication foci within discrete chromosome territories during interphase, we show that foci which are active during consecutive intervals of S phase are maintained as spatially adjacent neighbors throughout the cell cycle. Using extended DNA fibers, we demonstrate that this spatial continuity of replication foci correlates with the genetic continuity of adjacent replicon clusters along chromosomes. Finally, we used bioinformatic tools to compare the structure of DNA foci with DNA domains that are seen to replicate during discrete time intervals of S phase using genome-wide strategies. Data presented show that a major mechanism of S phase progression involves the sequential synthesis of regions of the genome because of their genetic continuity along the chromosomal fiber.     

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.     

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.     

Cdc45 limits replicon usage from a low density of preRCs in mammalian cells

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4-5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ～0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ～70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase.     

Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed “functional organization” of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1–28 and 111–179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH₂-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH₂ or the COOH termini of heterologous proteins.

We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.

     

Replication origins in Xenopus egg extract Are 5-15 kilobases apart and are activated in clusters that fire at different times

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [(3)H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5-15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5-10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.