Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences

Five distinct patterns of DNA replication have been identified during S-phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus. In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin. As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase. At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin. Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the BrdU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific alpha-satellite DNA sequences indicates that the alpha-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.     

Modification specifique des bandes Q et R par le 5-bromodeoxyuridine et l'actinomycine D

When introduced into human lymphocyte culture, 5-bromodeoxyuridine (BUdR) and actinomycin D (AMD) induce chromosome differentiation by lack of condensation of segments corresponding to Q-bands (BUdR) and R-bands (BUdR and AMD). The total amount of DNA per cell is not modified by these treatments. The non-condensed segments partly keep their properties of R- or Q-banding after heat treatment or staining with quinacrine mustard. On the other hand, they lose their properties after ASG treatment (G-bands), and emit modified fluorescence after staining with acridine orange. With heat treatment or QM staining, it seems that BUdR or AMD elongate the R or Q segments in several ways—homogeneous repartition or fragmentation of various types. On the other hand, this elongation seems homogeneous after Feulgen staining. This suggests that the relation between Feulgen-revealed DNA and substratum of the R- and Q-bands might not be direct.     

Traitements discontinus par le BrdU et coloration par l'acridine orange: obtention de marquages R,Q et intermëdiaires

Discontinued treatments with BudR at different periods of the cellular cycle produce various chromosome banding after staining with acridine orange. In particular, it is possible to observe R- or Q- or an intermediary banding, simply by varying the time of incorporation of BudR. This implies that the amount of AT or GC bases present locally in DNA is not directly responsible for the banding observed. Furthermore it appears that a precise correlation exists between replication and R- or Q-banding: the DNA located at each group of bands replicates either early (R-bands) or late (Q-bands). But these timings overlap towards the middle of phase S: if the treatment is given at that time, it is possible to observe aspects intermediary between Q and R.     

Decrease of DNA synthesis in amniotic fluid cells during the middle part of S-phase revealed by differential chromosome staining after incorporation of BrdU

The time sequence of DNA replication in partially synchronized human amniotic fluid cells has been analysed, employing BrdU incorporation techniques. —Regardless of the interval between removal of the methotrexate/uridine block and addition of BrdU during S-phase, the treatment results in an R-type replication pattern. Conversely, replacement of BrdU containing medium by another one with thymidine yields G-type replication patterns. A thymidine pulse during the first 4 h of S-phase results in R-type replication patterns; from 7–10 h after block removal it produces G-type pattern. In between, only faint red staining dots can be found indicating a marked decrease of replicational activity during the middle part of the S-phase.     

Mimosine Arrests the Cell Cycle after Cells Enter S-Phase

-Mimosine (β-N-[3-hydroxy-4-pyridone]-α-aminopropionic acid)—a rare amino acid derived fromMimosaandLeucaenaplants—arrests cells reversibly late during G1 phase or at the beginning of S-phase. If mimosine were to arrest cells immediately before S-phase, it would provide a superb tool for the investigation of the initiation of DNA synthesis. Therefore, we reexamined the point of action of mimosine. Mitotic HeLa cells were released into 200 μMmimosine and grown for 10 h to block them, before the cells were permeabilized and the amino acid removed by washing them thoroughly. On addition of the appropriate triphosphates, DNA synthesis—measured by the incorporation of [³²P]dTTP—began immediately; as it is known that such permeabilized cells cannot initiate DNA synthesis but can only resume elongating previously initiated chains, mimosine must arrest after DNA synthesis has begun. Moreover, cells grown in mimosine assembled functional replication factories—detected by immunolabeling after incorporation of biotin–dUTP—that were typical of those found early during S-phase. Disappointingly, it seems that mimosine—like aphidocolin—blocks only after cells enter S-phase.     

ReplicationDomain: a visualization tool and comparative database for genome-wide replication timing data

Background  

Eukaryotic DNA replication is regulated at the level of large chromosomal domains (0.5–5 megabases in mammals) within which replicons are activated relatively synchronously. These domains replicate in a specific temporal order during S-phase and our genome-wide analyses of replication timing have demonstrated that this temporal order of domain replication is a stable property of specific cell types.     

Control of DNA replication and spatial distribution of defined DNA sequences in salivary gland cells of Drosophila melanogaster

In dividing cells, each sequence replicates exactly once in each S-phase, but in cells with polytene chromosomes, some sequences may replicate more than once or fail to replicate during S-phase. Because of this differential replication, the control of replication in polytene cells must have some unusual features. Dennhöfer (1982a) has recently concluded that the total DNA content of the polytene cells of Drosophila salivary glands exactly doubles in each S-phase. This observation, along with previous studies demonstrating satellite underreplication in salivary gland cells, led us to consider the hypothesis that there is a doubling of DNA mechanism for the control of DNA replication in polytene cells. With this mechanism, a doubling of DNA content, rather than the replication of each sequence, would signal the end of a cycle of DNA replication. To test this hypothesis, we have reinvestigated the replication of several sequences (satellite, ribosomal, histone and telomere) in salivary gland cells using quantitative in situ hybridization. We find that underreplication of some sequences does occur. In addition we have repeated Dennhöfer's cytophotometric and labeling studies. In contrast to Dennhöfer, we find that the total DNA contents of nonreplicating nuclei do reflect this partial replication, in accord with Rudkin's (1969) result. We conclude that DNA replication in polytene cells is controlled by modifications of the mechanism operating in dividing cells, where control is sequence autonomous, and not by a doubling of DNA mechanism. — In situ hybridization to unbroken salivary gland nuclei reveals the distribution of specific sequences. As expected, satellite, histone and 5S sequences are usually in a single cluster. This rules out the possibility that sequences known to be underreplicated in chromosomal DNA exist as extrachromosomal copies. Telomere sequences are grouped into two to six clusters, as if the chromosome ends are partially but not completely paired in salivary gland nuclei.     

The behaviour of fragile X and other aberrations during recovery from low folate conditions

Whole blood from two mentally retarded fra-X brothers was grown in low folate medium where fra-X expression was enhanced. Bromodeoxyuridine was added to mitigate the low folate conditions and metaphases were sampled sequentially, and stained for replication banding, through one cell cycle of recovery. — The replication bands allowed detailed analysis of the cell cycle and the allocation of individual cells to precise sub-phases. Various classes of fra-X and all other types of chromosomal aberrations were scored in these classified cells. — The fra-X does not conform in morphology to any of the known simple chromatid intrachange types, which were often present within the same cells, but the subsequent fall in frequency once bromodeoxyuridine was added closely paralleled that of the conventional aberrations. — Normal folate level frequencies of fra-X are restored by the time early S-phase cells (subphase SkI) reach metaphase. When sub-phased cells are rearranged in true chronological sequence, there is a suggestion of a sudden fall in frequency between SkII–III (about 70% of the transit of S). This suggests that the critical point for low folate enhancement occurs in this region of the S-phase. This is somewhat earlier than the band-appearance distribution curve for Xq27 which lies within subphase SkIV.     

Chronology of the replication of sex chromosome bands in lymphocytes of normal subjects and patients]

The replication sequence of the bands carried by chromosomes X and Y has been studied in normal individuals and in patients with structural abnormalities of the X. By comparing the segment with that of the autosomal bands (which had been previously studied), it was shown that the normal early X replicates in early X-phase for its R-bands and in late S-phase for its Q bands. The late X replicates entirely in late S-phase, and the sequence of band replication is not as stringent as for the early X and the autosomes. The study of fourteen cases of anomalies of chromosome X in females showed the following: in balanced reciprocal X-autosome translocations the rearranged X most often replicates early and the normal X late. Both show a normal replication sequence of their bands. In non-balanced X-autosome translocations, inactivation of the autosome fragment attached to the AUTOSOME FRAGMENT ATTACHED TO THE X may take place. In Xq- or in ter rea (X;X) (pter;pter), band p22 has a delayed replication. In iso-Xor Xp-, the long-arm-band sequence of replication shows a variation comparable to that of the late X in fibroblasts. These replication modifications are likely to induce partial inactivations or changes in activity which correspond to the so-called position effect in Drosophila.     

Asynchrony in late replication between homologous autosomes in primary cultures of Chinese hamster fibroblasts

Asynchronies in late replication of the autosomal chromosome pair No. 5, and to some extent of pair No. 4, were found after thymidine pulse labeling cultures of partially synchronized Chinese hamster lung fibroblasts from nine to nine and a half hours and from nine and a half to ten hours after block removal. In contrast to this, no asynchrony could be detected in the replication of homologous autosomes after continuous labeling for the last two hours of the S-phase. — G-banding and C-banding revealed no differences between the homologous autosomes. — These findings indicate that besides the known form of asynchronous replication in mammalian cells during S-phase on the chromosomal level, there also exists an asynchronous replication between homologous autosomes of the same complement.     

Role of replication time in the control of tissue-specific gene expression.

Late-replicating chromatin in vertebrates is repressed. Housekeeping (constitutively active) genes always replicate early and are in the early-replicating R-bands. Tissue-specific genes are usually in the late-replicating G-bands and therein almost always replicate late. Within the G-bands, however, a tissue-specific gene does replicate early in those cell types that express that particular gene. While the condition of late replication may simply be coincident with gene repression, we review evidence suggesting that late replication may actively determine repression. As mammals utilize a developmental program to Lyonize (facultatively heterochromatinize) whole X chromosomes to a late-replicating and somatically heritable repressed state, similarly another program seems to Lyonize individual replicons. In frogs, all genes begin embryogenesis by replicating during a very short interval. As the developmental potency of embryonic cells becomes restricted, late-replicating DNA gradually appears. This addition to the repertoire of gene control--i.e., repression via Lyonization of individual replicons--seems to have evolved in vertebrates with G-bands being a manifestation of the mechanism.     

S-phase dependent forms of DNA - nuclear membrane complexes in HeLa cells.

DNA - nuclear membrane complexes were isolated from HeLa cells and examined by either zone sedimentation analysis or isopycnic centrifugation in sucrose/CsCl gradients. The data suggest that the complexes formed during the first 10 min of the S-phase remain as stable structures throughout the cell cycle. Other DNA - nuclear membrane complexes are formed at later times during replication. These later complexes appear as multiple species and the association of DNA and the nuclear membrane seems to be of a transient nature. Together, these results suggest that both the replicative origins and the replication points of the DNA are associated with the nuclear membrane. Although the complexes formed at the start of the S-phase and at later times during the S-phase appear to differ, these differences may provide them with the needed properties to serve as spatial organizers for the temporal regulation of DNA replication.     

Querscheibenspezifische Unterschiede in der3H-Thymidin-Markierung während der Larvenentwicklung vonChironomus thummi piger

In salivary gland chromosome II ofChironomus, region A2j-A3(d) replicates during the whole replication cycle.³H-thymidine is incorporated in this region for a longer time than in bands of greater DNA values (Hägele, 1970). However, no extra DNA is accumulated in A2j-A3(d). Therefore it was supposed that in addition to the duplication of structural DNA an extra DNA is synthesized which immediately disappears from the chromosome. In this report the attempt was made to test this hypothesis. — Using³H-thymidine, the autoradiographic patterns have been studied which occur in the salivary gland chromosomes II ofChironomus thummi piger at the end of a replication step. The probable order of their sequence has been established. At the mid phase of a replication step region A2j-A3(d) and a certain number of definite bands are labelled whereas at the very end of DNA synthesis only A2j-A3(d) shows labelling. It is demonstrated that this region replicates for a longer time than regions containing up to 3.8 times more DNA. Moreover in most cells³H-thymidine is incorporated in region A2j-A3(d) at the end of synthesis at a higher rate than in late replicating bands. In this region there exists a considerable difference in relative grain density within the same phase of a replication step. This difference cannot be found in other bands studied. — These labelling patterns occur in chromosomes of both young larvae (8–9 days old) and prepupae (15–17 days old) if the larvae are prepared immediately after incubation in the isotope. — However, if young larvae are incubated in³H-thymidine and then develop to prepupae in water free of isotope region, A2j-A3(d) is unlabelled at the end of a replication step in half of the cells studied. In the other half this region shows labelling but the relative grain density is markedly reduced. The labelling pattern of other bands is not changed. Therefore loss of radioactive DNA in A2j-A3(d) is of real occurrence. — This loss probably takes place within the replication steps 1 or 2 between young larvae and praepupae. In these replications the structural DNA and the extra DNA, newly synthesized in A2j-A3(d), are unlabelled. The extra DNA disappears immediately from the chromosome. If, by chance, an exchange takes place between newly synthesized unlabelled DNA chains of extra DNA and old labelled DNA, then loss of radioactive DNA would be the result.     

A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time.     

Variations in the length of S-phase related to the time cells are blocked at the G1-S interface

The inhibition of DNA synthesis with hydroxyurea or 5-fluorodeoxyuridine decreases the duration of S-phase of synchronously growing Chinese hamster cultures. — The observed drug effects are discussed in relation to an alteration of programmed DNA replication.     

Specific modification of Q and R bands by 5-bromodeoxyuridine and actinomycin D]

When introduced into human lymphocyte culture, 5-bromodeoxyuridine (BUdR) and actinomycin D (AMD) induce chromosome differentiation by lack of condensation of segments corresponding to Q-bands (BUdR) and R-bands (BUdR and AMD). The total amount of DNA per cell is not modified by these treatments. The non-condensed segments partly keep their properties of R- or Q-banding after heat treatment or staining with quinacrine mustard. On the other hand, they lose their properties after ASG treatment (G-bands), and emit modified fluorescence after staining with acridine orange. With heat treatment or QM staining, it seems that BUdR or AMD elongate the R or Q segments in several ways—homogeneous repartition or fragmentation of various types. On the other hand, this elongation seems homogeneous after Feulgen staining. This suggests that the relation between Feulgen-revealed DNA and substratum of the R- and Q-bands might not be direct.     

ARS replication during the yeast S phase

A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1. Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication. A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase. The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication. The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ. These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase. If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins.