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1.
Mcm10 (Dna43) is an essential protein for the initiation of DNA replication in Saccharomyces cerevisiae. Recently, we identified a human Mcm10 homolog and found that it is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner and that it binds chromatin exclusively during the S phase of the cell cycle. However, the precise roles that Mcm10 plays are still unknown. To study the localization dynamics of human Mcm10, we established HeLa cell lines expressing green fluorescent protein (GFP)-tagged Mcm10. From early to mid-S phase, GFP-Mcm10 appeared in discrete nuclear foci. In early S phase, several hundred foci appeared throughout the nucleus. In mid-S phase, the foci appeared at the nuclear periphery and nucleolar regions. In the late S and G phases, GFP-Mcm10 was localized to nucleoli. Although (2)the distributions of GFP-Mcm10 during the S phase resembled those of replication foci, GFP-Mcm10 foci did not colocalize with sites of DNA synthesis in most cases. Furthermore, the transition of GFP-Mcm10 distribution patterns preceded changes in replication foci patterns or proliferating cell nuclear antigen foci patterns by 30-60 min. These results suggest that human Mcm10 is temporarily recruited to the replication sites 30-60 min before they replicate and that it dissociates from chromatin after the activation of the prereplication complex. 相似文献
2.
Although the molecular enzymology of DNA replication is well characterised, how and why it occurs in discrete nuclear foci is unclear. Using fission yeast, we show that replication takes place in a limited number of replication foci, whose distribution changes with progression through S phase. These sites define replication factories which contain on average 14 replication forks. We show for the first time that entire foci are mobile, able both to fuse and re-segregate. These foci form distinguishable patterns during S phase, whose succession is reproducible, defining early-, mid- and late-S phase. In wild-type cells, this same temporal sequence can be detected in the presence of hydroxyurea (HU), despite the reduced rate of replication. In cells lacking the intra-S checkpoint kinase Cds1, replication factories dismantle on HU. Intriguingly, even in the absence of DNA damage, the replication foci in cds1 cells assume a novel distribution that is not present in wild-type cells, arguing that Cds1 kinase activity contributes to the spatio-temporal organisation of replication during normal cell growth. 相似文献
3.
Leonhardt H Rahn HP Weinzierl P Sporbert A Cremer T Zink D Cardoso MC 《The Journal of cell biology》2000,149(2):271-280
DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase. 相似文献
4.
The sequential organisation of replication foci during S phase in onion ( Allium cepa) and their relationship to the nuclear matrix were investigated. To discern their structural features and temporal firing sequence, immunodetection of 5-bromo-2'-deoxyuridine (BrdU) was carried out after in vivo feeding in synchronised cells released from a 14-h-long hydroxyurea block. Replication foci consisted of small replication granules, called replisomes, which clustered together. Analysis of synchronous binucleate cells that maintained in their two nuclei the specular symmetry of distribution of sister chromosomes in anaphase, showed that replication starts in small replication foci at the telomeric pole (pattern I), though the telomeres themselves formed large foci that were late-replicating. The rDNA replication foci (pattern II) also become replicated in early S phase. Replication of large foci, including the heterochromatin (IV), occurred in late S phase and finished at the centromeric nuclear pole (pattern V). Labelling of proliferating cell nuclear antigen (PCNA) in nuclear matrices, prepared from S-phase nuclei after extensive DNase digestion, demonstrated that replication foci were always stably anchored to the nuclear matrix. Thus, association with the nucleoskeleton is not exclusively mediated by the replicating or nascent DNA. The overlapping of patterns I, II and III in the nuclear matrix, in contrast to the results of BrdU localisation in nuclei, suggests that PCNA becomes associated with the nuclear matrix before the replication foci are operative, and remains bound during replication. 相似文献
5.
DNA polymerase epsilon (pol epsilon) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol epsilon was examined by indirect immunofluorescence, pol epsilon appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol epsilon foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol epsilon foci were present throughout mitosis and the G(1) phase of cycling cells. It is hypothesized from these observations that pol epsilon and PCNA have separate but associated functions early in S phase and that pol epsilon participates with PCNA in DNA replication late in S phase. 相似文献
6.
G. Banfalvi H. Tanke A. K. Raap J. Slats M. van der Ploeg 《Histochemistry and cell biology》1990,94(4):435-440
Summary DNA replication sites generally known as replicon domains were resolved as individual replication signals in interphase nuclei of permeabilized Chinese hamster ovary cells by immunofluorescent microscopy. Biotin-11-dUTP was utilized as a tool to label newly replicated DNA in permeable cells and to study the distribution of nascent DNA in pulselabel and in pulsechase experiments. Active sites of DNA replication were visualized in exponentially growing cells and in synchronized cultures throughout the S phase. Fluorescent images of replication sites were analyzed by standard fluorescense microscopy and in three dimensions by confocal laser scanning microscopy. The rapid increase in number of discrete foci of newly replicated DNA is an indication that DNA synthesis starts at limited number of sites in mammalian nuclei rather than at thousands of foci at the same time. 相似文献
7.
Colocalization of Baculovirus IE-1 and Two DNA-Binding Proteins, DBP and LEF-3, to Viral Replication Factories 总被引:10,自引:6,他引:4
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We have recently identified a DNA-binding protein (DBP) from the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) which can destabilize double-stranded DNA (V. S. Mikhailov, A. L. Mikhailova, M. Iwanaga, S. Gomi, and S. Maeda, J. Virol. 72:3107–3116, 1998). DBP was found to be an early gene product that was not present in budded or occlusion-derived virions. In order to characterize the localization of DBP during viral replication, BmNPV-infected BmN cells were examined by immunostaining and confocal microscopy with DBP antibodies. DBP first appeared as diffuse nuclear staining at 4 to 6 h postinfection (p.i.) and then localized to several specific foci within the nucleus at 6 to 8 h p.i. After the onset of viral DNA replication at around 8 h p.i., these foci began to enlarge and eventually occupied more than half of the nucleus by 14 h p.i. After the termination of viral DNA replication at about 20 h p.i., the DBP-stained regions appeared to break down into approximately 100 small foci within the nucleus. At 8 h p.i., the distribution of DBP as well as that of IE-1 or LEF-3 (two proteins involved in baculovirus DNA replication) overlapped well with that of DNA replication sites labeled with bromodeoxyuridine incorporation. Double-staining experiments with IE-1 and DBP or IE-1 and LEF-3 further confirmed that, between 8 and 14 h p.i., the distribution of IE-1 and LEF-3 overlapped with that of DBP. However, IE-1 localized to the specific foci prior to DBP or LEF-3 at 4 h p.i. In the presence of aphidicolin, an inhibitor of DNA synthesis, immature foci containing IE-1, LEF-3, and DBP were observed by 8 h p.i. However, the subsequent enlargement of these foci was completely suppressed, suggesting that the enlargement depended upon viral DNA replication. At 4 h p.i., the number of IE-1 foci correlated with the multiplicity of infection (MOI) between 0.4 and 10. At higher MOIs (e.g., 50), the number of foci plateaued at around 15. These results suggested that there are about 15 preexisting sites per nucleus which are associated with the initiation of viral DNA replication and assembly of viral DNA replication factories. 相似文献
8.
Essers J Theil AF Baldeyron C van Cappellen WA Houtsmuller AB Kanaar R Vermeulen W 《Molecular and cellular biology》2005,25(21):9350-9359
The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a "sliding clamp" that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair. 相似文献
9.
Visualization of double-stranded RNA in cells supporting hepatitis C virus RNA replication 总被引:3,自引:0,他引:3
The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for double-stranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescence-based hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells. 相似文献
10.
11.
Background
Eukaryotic cells seem unable to monitor replication completion during normal S phase, yet must ensure a reliable replication completion time. This is an acute problem in early Xenopus embryos since DNA replication origins are located and activated stochastically, leading to the random completion problem. DNA combing, kinetic modelling and other studies using Xenopus egg extracts have suggested that potential origins are much more abundant than actual initiation events and that the time-dependent rate of initiation, I(t), markedly increases through S phase to ensure the rapid completion of unreplicated gaps and a narrow distribution of completion times. However, the molecular mechanism that underlies this increase has remained obscure.Methodology/Principal Findings
Using both previous and novel DNA combing data we have confirmed that I(t) increases through S phase but have also established that it progressively decreases before the end of S phase. To explore plausible biochemical scenarios that might explain these features, we have performed comparisons between numerical simulations and DNA combing data. Several simple models were tested: i) recycling of a limiting replication fork component from completed replicons; ii) time-dependent increase in origin efficiency; iii) time-dependent increase in availability of an initially limiting factor, e.g. by nuclear import. None of these potential mechanisms could on its own account for the data. We propose a model that combines time-dependent changes in availability of a replication factor and a fork-density dependent affinity of this factor for potential origins. This novel model quantitatively and robustly accounted for the observed changes in initiation rate and fork density.Conclusions/Significance
This work provides a refined temporal profile of replication initiation rates and a robust, dynamic model that quantitatively explains replication origin usage during early embryonic S phase. These results have significant implications for the organisation of replication origins in higher eukaryotes. 相似文献12.
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 相似文献
13.
To study the dynamics of protein recruitment to DNA lesions, ion beams can be used to generate extremely localized DNA damage within restricted regions of the nuclei. This inhomogeneous spatial distribution of lesions can be visualized indirectly and rapidly in the form of radiation-induced foci using immunocytochemical detection or GFP-tagged DNA repair proteins. To analyze faster protein translocations and a possible contribution of radiation-induced chromatin movement in DNA damage recognition in live cells, we developed a remote-controlled system to obtain high-resolution fluorescence images of living cells during ion irradiation with a frame rate of the order of seconds. Using scratch replication labeling, only minor chromatin movement at sites of ion traversal was observed within the first few minutes of impact. Furthermore, time-lapse images of the GFP-coupled DNA repair protein aprataxin revealed accumulations within seconds at sites of ion hits, indicating a very fast recruitment to damaged sites. Repositioning of the irradiated cells after fixation allowed the comparison of live cell observation with immunocytochemical staining and retrospective etching of ion tracks. These results demonstrate that heavy-ion radiation-induced changes in subnuclear structures can be used to determine the kinetics of early protein recruitment in living cells and that the changes are not dependent on large-scale chromatin movement at short times postirradiation. 相似文献
14.
Three-dimensional analysis of DNA replication foci: a comparative study on species and cell type in situ 总被引:2,自引:2,他引:0
Chromatin morphology of interphase nuclei in most cell lines of quail (Coturnix coturnix japonica) and chick (Gallus gallus domesticus) embryos shows typical interspecies differences. This intrinsic marker has been used in quail/chick chimerisation experiments,
where also differences between cell types were noted. We asked whether similar differences between species and between cell
types could be observed in S phase nuclei in situ. In this report, we used bromodeoxyuridine (BrdU) pulse labelling and anti-BrdU
immunofluorescence to detect DNA replication foci in the nuclei of identified cells. In the central nervous system of 5- to
7-day-old quail and chick embryos, mesoderm-derived cells with strikingly different morphology and topographical distribution
were studied: endothelial, i.e. polarised cells forming continuous tubes, and macrophages, i.e. non-polarised, ameboid or
ramified individual cells. Using confocal microscopy, replication foci in the nuclei were assessed quantitatively and three-dimensional
visualisations were produced. We consistently observed that: (1) chick, but never quail, nuclei displayed completely confluent
replication sites, independent of cell type, and (2) macrophages, but not endothelial cells, had distinct perinucleolar replication
sites, independent of species. We thus demonstrate a new relationship between cell type and spatial arrangement of DNA replication
sites, and conclude that interspecies differences of chromatin distribution are conserved throughout S phase. Our results
strongly recommend that work done on nuclear structure in vitro should not be extrapolated without reservation to cells in
vivo.
Accepted: 5 January 2000 相似文献
15.
Nathana?l Hozé Myriam Ruault Carlo Amoruso Angela Taddei David Holcman 《Molecular biology of the cell》2013,24(11):1791-1800
Spatial and temporal behavior of chromosomes and their regulatory proteins is a key control mechanism in genomic function. This is exemplified by the clustering of the 32 budding yeast telomeres that form foci in which silencing factors concentrate. To uncover the determinants of telomere distribution, we compare live-cell imaging with a stochastic model of telomere dynamics that we developed. We show that random encounters alone are inadequate to produce the clustering observed in vivo. In contrast, telomere dynamics observed in vivo in both haploid and diploid cells follows a process of dissociation–aggregation. We determine the time that two telomeres spend in the same cluster for the telomere distribution observed in cells expressing different levels of the silencing factor Sir3 protein, limiting for telomere clustering. We conclude that telomere clusters, their dynamics, and their nuclear distribution result from random motion, aggregation, and dissociation of telomeric regions, specifically determined by the amount of Sir3. 相似文献
16.
17.
The distribution of chromatin within the mammalian nucleus is constrained by its organization into chromosome territories (CTs). However, recent studies have suggested that promiscuous intra- and inter-chromosomal interactions play fundamental roles in regulating chromatin function and so might define the spatial integrity of CTs. In order to test the extent of DNA mixing between CTs, DNA foci of individual CTs were labeled in living cells following incorporation of Alexa-488 and Cy-3 conjugated replication precursor analogues during consecutive cell cycles. Uniquely labeled chromatin domains, resolved following random mitotic segregation, were visualized as discrete structures with defined borders. At the level of resolution analysed, evidence for mixing of chromatin from adjacent domains was only apparent within the surface volumes where neighboring CTs touched. However, while less than 1% of the nuclear volume represented domains of inter-chromosomal mixing, the dynamic plasticity of DNA foci within individual CTs allows continual transformation of CT structure so that different domains of chromatin mixing evolve over time. Notably, chromatin mixing at the boundaries of adjacent CTs had little impact on the innate structural properties of DNA foci. However, when TSA was used to alter the extent of histone acetylation changes in chromatin correlated with increased chromatin mixing. We propose that DNA foci maintain a structural integrity that restricts widespread mixing of DNA and discuss how the potential to dynamically remodel genome organization might alter during cell differentiation. 相似文献
18.
The incC and korB genes of IncP-1 plasmid RK2 encode homologues of ubiquitous ParA and ParB partitioning proteins of bacterial plasmids and chromosomes. Using immunofluorescence microscopy, we found that KorB, which binds to 12 widely distributed sites on the genome, is located in symmetrically placed foci in cells containing IncP-1 plasmids. When maintained by the low-copy-number P7 replicon, an RK2 segment including incC, korB and the kla, kle and korC regions encodes an efficient partitioning system that gives a pattern of foci similar to RK2 itself. Symmetrical distribution of KorB foci correlates with segregational stability conferred by either the IncP-1 or P7 partitioning systems; KorB distribution follows plasmid distribution. In the absence of a second partitioning system, incC inactivation resulted in paired or clumped foci that were not symmetrically distributed. At a slow growth rate, position analysis of foci showed a cycle from one central focus to two foci (at one- and three-quarter positions) and back, and at a high growth rate it showed a cycle from two foci to four and back. This pattern fits with the plasmid being coupled to the replication zones in the cell and being moved to successively younger zones by active partitioning, indicating a tight association between replication and partitioning. 相似文献
19.
20.
It is still unclear what nuclear components support initiation of DNA replication. To address this issue, we developed a cell-free replication system in which the nuclear matrix along with the residual matrix-attached chromatin was used as a substrate for DNA replication. We found out that initiation occurred at late G1 residual chromatin but not at early G1 chromatin and depended on cytosolic and nuclear factors present in S phase cells but not in G1 cells. Initiation of DNA replication occurred at discrete replication foci in a pattern typical for early S phase. To prove that the observed initiation takes place at legitimate DNA replication origins, the in vitro synthesized nascent DNA strands were isolated and analyzed. It was shown that they were enriched in sequences from the core origin region of the early firing, dihydrofolate reductase origin of replication ori-beta and not in distal to the origin sequences. A conclusion is drawn that initiation of DNA replication occurs at discrete sub-chromosomal structures attached to the nuclear matrix. 相似文献