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.     

Description of a chromosome replication unit in individual prematurely condensed human S-phase chromosomes

Mammalian chromosome replication was studied by the aid of premature chromosome condensation (PCC). After induction of PCC the sites of DNA replication appear as “gaps” between condensed chromosomal regions. These condensed particles are unineme before and bineme after DNA replication. The two phases are due mainly to the unineme or bineme nature of the particles. During early S-phase almost all particles are unineme, during late S-phase they are bineme and there is only one transitory stage between these two main stages. Premature chromosome condensation was studied in detail on a specific human chromosome 22 which is marked by its heterochromatin constitution. This led to easy identification of these elements in S-phase PCC (S-PCC) preparations. For each stage of the S-phase there was a reproducible pattern of condensed chromosomal particles making up the whole chromosome. The number of these particles was rather limited and a complementary pattern was found in early versus late S-phase. The pattern of early S-PCC corresponded to the banding pattern of G-banded prometaphase chromosomes; the pattern of late S-PCC, to R-banded prometaphase chromosomes. Thus, “gaps” and condensed particles as observed after PCC induction are obviously homologous to chromosome replication units. Replication of constitutive heterochromatin occurred during the very late S-phase. During this stage PCC induction led to condensation of the heterochromatin into several small, highly fluorescent particles.     

DNA REPLICATION PATTERNS AND CHROMOSOMAL PROTEIN SYNTHESIS IN OPOSSUM LYMPHOCYTES IN VITRO

DNA replication patterns were determined in the autosomes and sex chromosomes of phytohemagglutinin-stimulated lymphocytes from the opossum (Didelphis virginiana) by employing thymidine-³H labeling and high-resolution radioautography. Opossum chromosomes are desirable experimental material due to their large size, low number (2n = 22), and morphologically distinct sex chromosomes. The autosomes in both sexes began DNA synthesis synchronously and terminated replication asynchronously. One female X chromosome synthesized DNA throughout most of the S phase. Its homologue, however, began replication approximately 3.5 hr later. The two X's terminated DNA synthesis synchronously, slightly later than the autosomes. This form of late replication, in which one X chromosome begins DNA synthesis later than its homologue but completes replication at the same time as its homologue, is apparently unique in the opossum. The male X synthesized DNA throughout S while the Y chromosome exhibited late-replicating characteristics. The two sex chromosomes completed synthesis synchronously, slightly later than the autosomes. Grain counts were performed on all chromosomes to analyze trends in labeling intensity at hourly intervals of S. By analyzing the percent of labeled mitotic figures on radioautographs at various intervals after introduction of arginine-³H, chromosomal protein synthesis was found not to be restricted to any portion of interphase but to increase throughout S and into G₂.     

Early and late DNA replication in respectively condensed and decondensed heterochromatin ofAllium carinatum

Summary The structure and ultrastructure of nuclei in the S period and other phases of the mitotic cell cycle have been studied in semi- and ultrathin sections of root tips ofAllium carinatum. Significant structural differences have been found and classified by means of DNA measurements by scanning photometry of Feulgen-stained squash preparations. In G₁ and early S (S₁ and S₂) the euchromatin forms small, compact and electron-dense patches, while the heterochromatin is condensed into a number of chromocenters of the same electron-density as the euchromatin. In middle S (S₃) the euchromatic elements become larger and more thread-like. In late S (S₄) the euchromatin appears in the form of thick and uniform strands as in G₂, and the heterochromatin decondenses into strands of the same, or a little higher, diameter, as the euchromatin. DNA replication starts in the condensed heterochromatin (S₁, becomes shifted to euchromatin (S₂), continues over both eu- and heterochromatin during middle S (S₃), and is restricted to decondensed heterochromatin in late S (S₄). Quantitative data of various nuclear parameters are given for the different stages. The results are discussed in relation to the species-specific nuclear ultrastructure, its molecular basis, and its variation during the mitotic interphase, as well as with respect to the timing and structural expression of DNA replication.     

DNS-Replikation und Morphologie der X-Chromosomen während der Syntheseperiode bei Microtus agrestis

X-chromosome behaviour of female Microtus agrestis in interphase nuclei with and without large chromocenters was examined in cultured epithelial and fibroblast cells. By means of pulse-labeling, the configuration of the X-chromosomes in these nuclei can be illustrated; staining with pararosaniline-methylgreen seems to be most suitable. According to the replication behaviour, three types of chromatin can be discerned in the X-chromosomes: early replicating euchromatin, late replicating sex chromatin, and very late replicating heterochromatin. In fibroblasts only the sex chromatin forms a single, small chromocenter; in epithelial cells both the sex chromatin and the remaining heterochromatin form large chromocenters. Both types of heterochromatin replicate their DNA in the condensed state. It seems likely that the late replicating segments of the X-chromosomes (sex chromatin and remaining heterochromatin) are condensed in every cell, but they may not always be configurated or even visible as typical chromocenters; these segments could be distributed over a wide range of compact to more or less diffuse superstructures.     

Early replicating DNA in heterochromatin ofPlagiochila ovalifolia (Liverworts)

The timing of DNA replication of heterochromatin in malePlagiochila ovalifolia was investigated by the use of³H-thymidine autoradiography. The estimated duration of the mitotic cycle was as follows: S period, 19 hr: G₂+prophase, 10 hr; G₁+meta-, ana-, telophase, 5 hr; total mitotic cycle, 34 hr. The first appearance of silver grains over the chromosomes was observed at 8 hr after the beginning of pulse labelling at which time the silver grains were only over the euchromatic regions, not over the heterochromatic regions. This labelling pattern was also observed at 10 to 15 hr. The heterochromatic regions having more grains than the euchromatic regions were observed at 20 to 25 hr. These results show that the DNA of the heterochromatin of this species is replicated earlier than the euchromatin.     

Chromatin structure during the prereplicative phases in the life cycle of mammalian cells

The object of this study was to determine the kinetics of chromosome decondensation during the G₁ period of the HeLa cell cycle. HeLa cells synchronized in the G₁ period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N₂O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G₁ period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G₁ period, when DNA synthesis is initiated.     

Initiation of DNA synthesis in the prematurely condensed chromosomes of G1 cells

The objective of this study was to investigate whether G1 cells could enter S phase after premature chromosome condensation resulting from fusion with mitotic cells. HeLa cell synchronized in early G1, mid-G1, late G1, and G2 and human diploid fibroblasts synchronized in G0 and G1 phases were separately fused by use of UV-inactivated Sendai virus with mitotic HeLa cells. After cell fusion and premature chromosome condensation, the fused cells were incubated in culture medium containing Colcemid (0.05 micrograms/ml) and [3H]thymidine ([3H]ThdR) (0.5 microCi/ml; sp act, 6.7 Ci/mM). At 0, 2, 4, and 6 h after fusion, cell samples were taken to determine the initation of DNA synthesis in the prematurely condensed chromosomes (PCC) on the basis of their morphology and labeling index. The results of this study indicate that PCC from G0, G1, and G2 cells reach the maximum degree of compaction or condensation at 2 h after PCC induction. In addition, the G1-PCC from normal and transformed cells initiated DNA synthesis, as indicated by their "pulverized" appearance and incorporation of [3H]ThdR. Further, the initiation of DNA synthesis in G1-PCC occurred significantly earlier than in the mononucleate G1 cells. Neither pulverization nor incorporation of label was observed in the PCC of G0 and G2 cells. These findings suggest that chromosome decondensation, although not controlling the timing of a cell's entry into S phase, is an important step for the initiation of DNA synthesis. These data also suggest that the entry of a S phase may be regulated by cell cycle phase-specific changes in the permeability of the nuclear envelope to the inducers of DNA synthesis present in the cytoplasm.     

Differential reactivity in mammalian chromosomes

Leucocyte cultures were treated with both ³H-thymidine and low temperature. Leucocyte cells were pulse labeled with ³H-thymidine for 15 to 20 minutes, and then placed in nonisotopic medium for 0, 1, 2, 3 and 4 hours respectively. Each culture was immediately treated with low temperature at 0–3° C for 24 hours. No metaphase chromosome were labeled at 0 and 1 hour after reincubation. Labeled metaphases were first observed after 2 hours of reincubation (3.9%); they increased after 3 hours (57%) and 4 hours of reincubation (39%). Labeled anaphases or telophases were also detectable in increasing proportions after 4 hours. Cell division proceeds very slowly through metaphase at low temperature. After labeling in the final 15 to 20 minutes of the S-period, one X-chromosome usually showed the late-replicating pattern. Label was found in the special segments of the X-chromosomes, X_E–a, X_L–a and X_L–b. Late-replicating regions in autosomes coincide more or less with the special segments. Differential reactivity in human chromosomes by low temperature was suggested to take place during the final part of G₂ after DNA synthesis.     

Cytological studies of heterochromatin function in the Drosophila melanogaster male: autosomal meiotic pairing

In Drosophila melanogaster it is now documented that the different satellite DNA sequences make up the majority of the centromeric heterochromatin of all chromosomes. The most popular hypothesis on this class of DNA is that satellite DNA itself is important to the pairing processes of chromosomes. Evidence in support of such a hypothesis is, however, circumstantial. This hypothesis has been evaluated by direct cytological examination of the meiotic behaviour of heterochromatically and/or euchromatically rearranged autosomes in the male. It was found that neither substantial deletions nor rearrangements of the autosomal heterochromatin cause any disruption of meiotic pairing. Autosomal pairing depends on homologs retaining sufficient euchromatic homology. This is the first clear demonstration that the highly repeated satellite DNA sequences in the heterochromatin of the second, third and fourth chromosomes are not important in meiotic pairing, but rather that some euchromatic homology in the autosomes is essential to ensure a regular meiotic process. These results on the autosomes, when taken in conjunction with our previous studies on sex chromosome pairing, clearly indicate that satellite DNA is not crucial for male meiotic chromosome pairing of any member of the D. melanogaster genome.     

Cytogenetic replication studies with short thymidine pulses in bromodeoxyuridine-substituted chromosomes of different mouse cell lines

Summary In normal diploid fibroblasts of the mouse, 3T3-, SV-3T3-, and Meth A-cells, the chromosome replication patterns were studied by a bromodeoxyuridine (BrdU)-labelling technique. SV-3T3 is a subline of 3T3 transformed by SV 40 and Meth A is a permanent cell line from Balb c transformed by methylcholanthrene. The use of 1 h thymidine pulses permits high resolution of the S-phase after partial synchronization of the cells at G₁/S in an otherwise BrdU-substituted S-phase. It could be shown that the autosomal heterochromatin of the mouse (Mus musculus) starts replication during the early S-phase (R-band replication), continues while R-band chromatin finishes, and still replicates when G-band chromatin starts. The heterochromatin finishes before the majority of G-bands have been replicated. There is no fundamental difference in the course of chromosome replication between the different cell lines studied here. It is concluded that there are no obligate changes in the course of the S-phase linked to the process of transformation.     

A correlation between radiation sensitivity and initial chromatid breaks in cancer cell lines revealed by Calyculin A-induced premature condensation

Three human malignancy cell lines were irradiated with ⁶⁰Co γ-rays. Initial chromatid breaks were measured by using the chemically induced premature chromosome condensation technique. Survival curves of cells exposed to gamma rays was linear-quadratic while the efficiency of Calyculin A in inducing PCC of G₂ PCC was about five times more than G₁ PCC. A dose-dependent increase in radiation-induced chromatid/isochromatid breaks was observed in G₁ and G₂ phase PCC and a nearly positive linear correlation was found between cell survival and chromatin breaks. This study implies that low LET radiation-induced chromatid/isochromatid breaks can potentially be used to predict the radiosensitivity of tumor cells either in in vitro experimentation or in in vivo clinical radiotherapy.     

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with ³H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.     

Atypical heterochromatin organization and replication are rapidly acquired by somatic cells following fusion-mediated reprogramming by mouse ESCs

We recently reported that mouse embryonic stem cells (ESCs) in S/G₂ are more efficient at reprogramming somatic cells than ESCs at other stages of the cell cycle. We also provided evidence that DNA replication is induced in the nuclei of somatic partners upon fusion with ESC partners, and showed that this was critical for their conversion toward a pluripotent state.¹ Here we have used counterflow centrifugal elutriation to enrich for ESCs at different cell cycle phases, so as to examine in detail the properties of S/G₂ phase cells. This revealed that the replication and organization of DAPI-intense heterochromatin in ESCs is unusual in two respects. First, replication of heterochromatin occurred earlier during S phase and was associated with precocious H3S10 phosphorylation. Second, heterochromatin protein 1 α (HP1α), which invariably marks DAPI-intense and H3K9me3-enriched pericentromeric domains in mouse somatic cells,² was not necessarily associated with these H3K9me3-enriched domains in undifferentiated ESCs. These data, which complement recent replication timing³ and electron spectroscopic imaging (ESI) analyses,⁴ suggest that heterochromatin is atypical in ESCs. Interestingly, as these unusual features were rapidly acquired by somatic nuclei upon ESC fusion-mediated reprogramming, our results suggest that fundamental changes in cell cycle structure and heterochromatin dynamics may be important for conferring pluripotency.     

Chromosomes and DNA of Mus: Terminal DNA synthetic sequences in three species

The DNA replication patterns of the terminal S phase of three species of Mus were analyzed by tritiated thymidine autoradiography. The centromeric heterochromatin of M. fulvidiventris is the latest component to finish DNA synthesis. The Y chromosome finishes replication earlier than the centromeric heterochromatin. The centromeric heterochromatin of M. musculus, on the other hand, is not the latest component to finish DNA synthesis. At the very late S phase, grains are found in the euchromatic arms instead of the heterochromatic areas. The hot X and the hot Y can be identified in the majority of, but not all, cases. The heterochromatic short arms of the autosomes in M. dunni finish DNA replication earlier than many areas in the euchromatic long arms and the heterochromatin of the sex chromosomes. This indicates that in M. dunni there are at least two types of heterochromatin. The late-replicating zones in the euchromatic long arms are distinctly banded. This banded grain pattern can be seen in all Mus species observed, but in M. dunni it is most exaggerated. Late-replicating chromosome segments can be demonstrated also by 2+ cycles of BUdR incorporation and Giemsa staining.     

Chronology and pattern of replication in the bone marrow chromosomes of Gallus domesticus

The duration of the cell generation, the chronology, and the pattern of chromosome duplication was studied in the bone marrow of Gallus domesticus. The duration of the phases of the cell cycle is: cell generation 17.5 hours, S period 9 hours. G₂ period plus prophase stage 2.5 hours, G₁ period 6 hours. Chromosome replication begins at many sites. During middle S it extends to the whole complement and finally finishes in small, late replicating regions of the macrochromosomes. Interchromosomal asynchrony of duplication at the initiation or at the end of the S period was not observed. Z-chromosomes begin and finish DNA synthesis synchronously with the other macrochromosomes. The W-chromosome in females is the last microchromosome to finish replication. However it ends DNA synthesis at about the same time as the macrochromosomes. Similarities and differences between chromosome replication in Aves and Mammalia are considered.     

SET8 plays a role in controlling G1/S transition by blocking lysine acetylation in histone through binding to H4 N-terminal tail

We report evidence suggesting that methyltransferase SET8 plays a novel role in regulating cell cycle by suppressing DNA replication through histone binding. First, the distribution of SET8 is strongly cell cycle-dependent. SET8 was concentrated in the nucleus during G₁ and G₂ phases, and was excluded from the nucleus during S phase. Second, at G₁/S transition, SET8 was degraded through ubiquitination via SCF/Skp2. Third, it was evident that the SET8 binds to the H4 N-terminal tail (H4NT) and blocks the acetylation of lysine residues K5, K8 and K12 of histone H4 during G₁. Such a blockage can hinder DNA replication. Fourth, SET8 binds to hypoacetylated but not hyperacetylated H4NT. Finally, overexpressing the histone-binding domain of SET8 appeared to suppress DNA replication and arrest the cell cycle before the G₁/S transition. Taken together, these findings suggest that SET8 can be a negative regulator of DNA replication and the destruction of SET8 is required for the onset of S phase.     

Chromosomes with Two Intact Axial Cores Are Induced by G2 Checkpoint Override: Evidence That DNA Decatenation Is not Required to Template the Chromosome Structure

Here we report that DNA decatenation is not a physical requirement for the formation of mammalian chromosomes containing a two-armed chromosome scaffold. 2-aminopurine override of G₂ arrest imposed by VM-26 or ICRF-193, which inhibit topoisomerase II (topo II)–dependent DNA decatenation, results in the activation of p34^cdc2 kinase and entry into mitosis. After override of a VM-26–dependent checkpoint, morphologically normal compact chromosomes form with paired axial cores containing topo II and ScII. Despite its capacity to form chromosomes of normal appearance, the chromatin remains covalently complexed with topo II at continuous levels during G₂ arrest with VM-26. Override of an ICRF-193 block, which inhibits topo II–dependent decatenation at an earlier step than VM-26, also generates chromosomes with two distinct, but elongated, parallel arms containing topo II and ScII. These data demonstrate that DNA decatenation is required to pass a G₂ checkpoint, but not to restructure chromatin for chromosome formation. We propose that the chromosome core structure is templated during interphase, before DNA decatenation, and that condensation of the two-armed chromosome scaffold can therefore occur independently of the formation of two intact and separate DNA helices.