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1.

Background

In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM). There is varied evidence indicating that DNA replication occurs in replication factories organized upon the NM and that DNA loops may correspond to the actual replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but they synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration in vivo. We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.

Results

In the present work we tracked down the movement relative to the NM of DNA sequences located at different points within such five structural DNA loops during the S phase and after the return to cellular quiescence during liver regeneration. Our results indicate that looped DNA moves sequentially towards the NM during replication and then returns to its original position in newly quiescent cells, once the liver regeneration has been achieved.

Conclusions

Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated. These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.  相似文献   

2.
3.
Kitamura E  Blow JJ  Tanaka TU 《Cell》2006,125(7):1297-1308
Faithful DNA replication ensures genetic integrity in eukaryotic cells, but it is still obscure how replication is organized in space and time within the nucleus. Using timelapse microscopy, we have developed a new assay to analyze the dynamics of DNA replication both spatially and temporally in individual Saccharomyces cerevisiae cells. This allowed us to visualize replication factories, nuclear foci consisting of replication proteins where the bulk of DNA synthesis occurs. We show that the formation of replication factories is a consequence of DNA replication itself. Our analyses of replication at specific DNA sequences support a long-standing hypothesis that sister replication forks generated from the same origin stay associated with each other within a replication factory while the entire replicon is replicated. This assay system allows replication to be studied at extremely high temporal resolution in individual cells, thereby opening a window into how replication dynamics vary from cell to cell.  相似文献   

4.
Nuclear DNA is looped by attachment to a matrix or cage. As this cage is the site of DNA synthesis, sequences in the loops must attach before they are replicated. We have tested whether sequences which initiate replication are usually out in the loop and attach only during S phase or whether they are attached but quiescent during most of the cell-cycle. Sequences which permit plasmids to replicate autonomously in yeast cells (ARS's) are strong candidates for initiating sequences. Four different human ARS's all map remote from attachment points to the HeLa nuclear cage. In addition a potential terminus of replication is also remote from the cage. We conclude that sequences involved in initiation are usually out in the loop and that DNA synthesis is initiated by their attachment.  相似文献   

5.
6.
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.

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7.
Eukaryotic DNA replication initiates at multiple origins. In early fly and frog embryos, chromosomal replication is very rapid and initiates without sequence specificity. Despite this apparent randomness, the spacing of these numerous initiation sites must be sufficiently regular for the genome to be completely replicated on time. Studies in various eukaryotes have revealed that there is a strict temporal separation of origin "licensing" prior to S phase and origin activation during S phase. This may suggest that replicon size must be already established at the licensing stage. However, recent experiments suggest that a large excess of potential origins are assembled along chromatin during licensing. Thus, a regular replicon size may result from the selection of origins during S phase. We review single molecule analyses of origin activation and other experiments addressing this issue and their general significance for eukaryotic DNA replication.  相似文献   

8.
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.  相似文献   

9.
We have investigated the attachment of the DNA to the nuclear matrix during the division cycle of the plasmodial slime mold Physarum polycephalum. The DNA of plasmodia was pulse labelled at different times during the S phase and the label distribution was studied by graded DNase digestion of the matrix-DNA complexes prepared from nuclei isolated by extraction with 2 M NaCl. Pulse labelled DNA was preferentially recovered from the matrix bound residual DNA at any time of the S phase. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the G2 phase and the subsequent S phase. The occurrence of the pulse label in the matrix associated DNA regions was transiently elevated at the onset of the subsequent S phase. Label incorporated at the end of the S phase was located at DNA regions which, in the G2 phase, were preferentially released from the matrix by DNase treatment. From the results and previously reported data on the distribution of attachment sites it can be concluded that origins of replicons or DNA sites very close to them are attached to the matrix during the entire nuclear cycle. The data further indicate that initiations of DNA replication occur at the same origins in successive S phases. Replicating DNA is bound to the matrix, in addition, by the replication fork or a region close to it. This binding is loosened after completion of the replication.  相似文献   

10.
Studies of DNA replication associated with the nuclear matrix have led to a radically new view of replication at the macroscopic level. It is proposed that individual replicons and their associated replicational assemblies (replisomes) are clustered together during active replication by attachment to the nuclear matrix at special sites termed 'clustersomes'. Direct visualization of replication sites in permeabilized cells by fluorescence microscopy following biotin-11-dUTP incorporation provides support for this model. Discrete replication granules are observed with sizes and numbers consistent with each granule being a site of replicon cluster synthesis. Distinct patterns of these sites are seen in different periods of S-phase. Both the individual granules and their early and late S-phase dependent patterns are strikingly maintained following extraction of the cells for in situ nuclear matrix structures. Similar results were obtained when probing in vivo sites of replication following incorporation of 5-bromodeoxyuridine. The three-dimensional organization of these replicational granules (clustersomes) is studied using confocal light microscopy and an appropriate multidimensional image analysis system.  相似文献   

11.
12.
We have investigated the possible relationship between replicons and chromatin loops during Xenopus development. In early embryos, replication of the ribosomal RNA genes (rDNA) can initiate at apparently any sequence. Nevertheless, the need for a regular spacing of replication origins suggests that some periodic chromatin folding might dictate which sites are actually used for initiation. After the midblastula transition, replication initiation is restricted to the rDNA intergenic spacers. A remodeling of chromatin folding could account for this change in origin usage. Here, it is reported that nuclear matrix anchorage of the Xenopus rDNA occurs at multiple, apparently random sequences, throughout embryonic development as well as in adult cells. In vitro matrix rebinding assays confirmed the lack of specific anchoring sequences in the rDNA, before as well as after specific replication origins are established. Thus, no change in loop attachment sites could explain the change in origin usage at this locus. Nonspecific loop anchorage was a special feature of the rDNA locus, since the same nuclear matrices were able selectively to bind the scaffold attachment region (SAR) of the Drosophila histone gene cluster in vitro. Blastula and gastrula nuclear matrices bound a higher amount of SAR sequences than matrices from later stages or adult cells. This developmental change in SAR binding might explain the increase in size of the bulk of genomic DNA loops that occurs after the gastrula stage. However, no change in chromatin loop organization that could explain the midblastula stage transition from small to large replicons was observed. Received: 15 January 1998; in revised form: 4 March 1998 / Accepted: 9 March 1998  相似文献   

13.
In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM). DNA loops are operationally classified in structural and facultative. Varied evidence indicates that DNA replication occurs in replication foci organized upon the NM and that structural DNA loops may correspond to the replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration. Using this model we have previously determined that the DNA loops corresponding to a gene-rich genomic region move in a sequential fashion towards the NM during replication and then return to their original configuration in newly quiescent cells, once liver regeneration has been achieved. In the present work we determined the organization into structural DNA loops of a gene-poor region centered on c-myc and tracked-down its movement at the peak of S phase and after the return to cellular quiescence during and after liver regeneration. The results confirmed that looped DNA moves towards the NM during replication but in this case the configuration of the gene-poor region into DNA loops becomes reorganized and after replication only the loop containing c-myc resembles the original in the control G0 hepatocytes. Our results suggest that the local chromatin configuration around potentially active genes constraints the formation of specific structural DNA loops after DNA replication, while in non-coding regions the structural DNA loops are only loosely determined after DNA replication by structural constraints that modulate the DNA-NM interactions.  相似文献   

14.
O Hyrien  M Mchali 《The EMBO journal》1993,12(12):4511-4520
We have analysed the replication of the chromosomal ribosomal DNA (rDNA) cluster in Xenopus embryos before the midblastula transition. Two-dimensional gel analysis showed that replication forks are associated with the nuclear matrix, as in differentiated cells, and gave no evidence for single-stranded replication intermediates (RIs). Bubbles, simple forks and double Ys were found in each restriction fragment analysed, showing that replication initiates and terminates without detectable sequence specificity. Quantification of the results and mathematical analysis showed that the average rDNA replicon replicates in 7.5 min and is 9-12 kbp in length. This time is close to the total S phase duration, and this replicon size is close to the maximum length of DNA which can be replicated from a single origin within this short S phase. We therefore infer that (i) most rDNA origins must be synchronously activated soon in S phase and (ii) origins must be evenly spaced, in order that no stretch of chromosomal DNA is left unreplicated at the end of S phase. Since origins are not specific sequences, it is suggested that this spatially and temporally concerted pattern of initiation matches some periodic chromatin folding, which itself need not rely on DNA sequence.  相似文献   

15.
16.
Is the nuclear matrix the site of DNA replication in eukaryotic cells?   总被引:1,自引:0,他引:1  
Four types of experiment were carried out to test the recently proposed model of matrix-bound replication in eukaryotic cells. In experiments with pulse-labelling we found preferential association of newly replicated DNA with the matrix only when the procedure for isolation includes first high-salt treatment of isolated nuclei and then digestion with nucleases, or when prior to digestion the nuclei have been stored for a prolonged time. In both cases, however, evidence was found that this preferential association is due to a secondary, artifactual binding of the newly replicated chromatin region to the matrix elements. Pulse-chase experiments and experiments with continuous labelling were carried out to answer the question whether during replication the DNA is reeled through the replication complexes, i.e., whether newly replicated DNA is temporarily or permanently associated with the matrix. The results showed that at that time the matrix DNA does not move from its site of attachment. Since, according to the model of matrix-bound replication, the forks are assumed to be firmly anchored to high-salt resistant proteinaceous matrix structures, the chromatin fragments isolated with endonuclease not recognizing newly replicated DNA and purified by sucrose gradient centrifugation should be free of replication intermediates. The electronmicroscopic analysis of such fragments revealed the existence of intact replication micro-bubbles. Moreover, the fragments with replication configurations appeared as smooth chromatin fibres not attached to elements characteristic for the matrix. All these experiments suggest that the nuclear skeleton is not a native site of DNA replication in eukaryotic cells.  相似文献   

17.
During S-phase of the cell cycle, chromosomal DNA is replicated according to a complex replication timing program, with megabase-sized domains replicating at different times. DNA fibre analysis reveals that clusters of adjacent replication origins fire near-synchronously. Analysis of replicating cells by light microscopy shows that DNA synthesis occurs in discrete foci or factories. The relationship between timing domains, origin clusters and replication foci is currently unclear. Recent work, using a hybrid Xenopus/hamster replication system, has shown that when CDK levels are manipulated during S-phase the activation of replication factories can be uncoupled from progression through the replication timing program. Here, we use data from this hybrid system to investigate potential relationships between timing domains, origin clusters and replication foci. We suggest that each timing domain typically comprises several replicon clusters, which are usually processed sequentially by replication factories. We discuss how replication might be regulated at different levels to create this complex organisation and the potential involvement of CDKs in this process.  相似文献   

18.
Localization of SV40 genes within supercoiled loop domains   总被引:18,自引:4,他引:14       下载免费PDF全文
Recent studies indicate that eukaryotic DNA is organized into supercoiled loop domains. These loops appear to be anchored at their bases to an insoluble nuclear skeleton or matrix. Most of the DNA in the loops can be released from the matrix by nuclease digestion; the residual DNA remaining with the nuclear matrix represents sequences at the base of the loops, and possibly other sequences which are intimately associated with the nuclear matrix for other reasons. Using a quantitative application of the Southern blotting technique, we have found this residual DNA from SV40 infected 3T3 cells to be enriched in SV40 sequences, indicating that they reside near matrix-DNA attachment points. An enrichment of 3-7 fold relative to total cellular DNA, was found in each of three different lines of SV40 infected 3T3 cells. Control experiments with globin genes showed no such enrichment in this residual matrix DNA. This sequence specificity suggests that the spatial organization of DNA sequences within loops may be related to the functionality of these sequences within the cell.  相似文献   

19.
We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.  相似文献   

20.
Origins of replication and gene regulation   总被引:13,自引:0,他引:13  
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