scholarly journals Radiation down-regulates replication origin activity throughout the S phase in mammalian cells

1999 ◽  
Vol 27 (3) ◽  
pp. 803-809 ◽  
Author(s):  
J. M. Larner ◽  
H. Lee ◽  
P. A. Dijkwel ◽  
R. D. Little ◽  
C. L. Schildkraut ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Replication Origin ◽  
S Phase

Fish oil slows S phase progression and may cause upstream shift of DHFR replication origin ori-β in CHO cells

2002 ◽  
Vol 283 (4) ◽  
pp. C1009-C1024 ◽  
Author(s):  
Nawfal W. Istfan ◽  
Zhi-Yi Chen ◽  
Sybille Rex
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Replication Origin ◽  
Dna Analysis ◽  
Corn Oil ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
S Phase ◽  
Replication Initiation ◽  
Temporal Organization

Fish oils (FOs) have been noted to reduce growth and proliferation of certain tumor cells, effects usually attributed to the content of polyunsaturated fatty acids of the n–3 family, which are thought to modulate cellular signaling pathways. We investigated the influence of FO on cell cycle kinetics of cultured Chinese hamster ovary cells. Exponentially growing cells were labeled with 5-bromo-2′-deoxyuridine (BrdU) and analyzed by flow cytometry after 5-day treatment with exogenous fat. Bivariate BrdU-DNA analysis indicated slower progression through S phase and thus longer S phase duration time in FO- but not corn oil-treated or control cells. We hypothesize that FO treatment might interfere with spatial/temporal organization of replication origins. Therefore, we mapped the well-characterized replication origin ori-β downstream of the dihydrofolate reductase gene with the nascent strand length assay. Three DNA marker segments with known positions relative to this origin were amplified by PCR. By quantitatively assessing DNA length of the fragments in all fractions containing these markers, the location of ori-β was established. In control or corn oil-treated cells, the location of ori-β was consistent with previous studies. However, in FO-treated cells, DNA replication appears to start from a new site located farther upstream from ori-β, suggesting a different replication initiation pattern. This study suggests novel mechanism(s) by which fats affect cell proliferation and DNA replication in mammalian cells.

scholarly journals Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yongzheng Li ◽  
Boxin Xue ◽  
Mengling Zhang ◽  
Liwei Zhang ◽  
Yingping Hou ◽  
...  
Keyword(s):  
Dna Replication ◽  
Structural Dynamics ◽  
Replication Origin ◽  
High Efficiency ◽  
S Phase ◽  
Chromatin Domain ◽  
Replication Origins ◽  
Replication Efficiency ◽  
Topologically Associating Domains ◽  
Epigenetic Signatures

Abstract Background Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined. Results We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired. Conclusion Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics.

Overexpression of kin17 protein disrupts nuclear morphology and inhibits the growth of mammalian cells

1999 ◽  
Vol 112 (19) ◽  
pp. 3215-3224 ◽  
Author(s):  
P. Kannouche ◽  
J.F. Angulo
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Reca Protein ◽  
S Phase ◽  
Expression Vectors ◽  
Nuclear Morphology ◽  
Functional Domain ◽  
Genetic Response ◽  
Cycle Progression

UVC or ionizing radiation of mammalian cells elicits a complex genetic response that allows recovery and cell survival. Kin17 gene, which is highly conserved among mammals, is upregulated during this response. Kin17 gene encodes a 45 kDa protein which binds to DNA and presents a limited similarity with a functional domain of the bacterial RecA protein. Kin17 protein is accumulated in the nucleus of proliferating fibroblasts and forms intranuclear foci. Using expression vectors, we show that overexpression of kin17 protein inhibits cell-cycle progression into S phase. Our results indicate that growth inhibition correlates with disruption of the nuclear morphology which seems to modify the intranuclear network required during the early steps of DNA replication. We report that a mutant encoding a protein deleted from the central domain of kin17 protein enhanced these effects whereas the deletion of the C-terminal domain considerably reduced them. These mutants will be used to elucidate the molecular mechanism by which kin17 protein alters cell growth and DNA replication.

scholarly journals High-throughput analysis of DNA replication in single human cells reveals constrained variability in the location and timing of replication initiation

2021 ◽  
Author(s):  
Dashiell J Massey ◽  
Amnon Koren
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Replication Timing ◽  
S Phase ◽  
Human Cells ◽  
Replication Initiation ◽  
High Throughput Analysis ◽  
Timing Variability ◽  
Fixed Set ◽  
Fixed Order

DNA replication occurs throughout the S phase of the cell cycle, initiating from replication origin loci that fire at different times. Debate remains about whether origins are a fixed set of loci used across all cells or a loose agglomeration of potential origins used stochastically in individual cells, and about how consistent their firing time during S phase is across cells. Here, we develop an approach for profiling DNA replication in single human cells and apply it to 2,305 replicating cells spanning the entire S phase. The resolution and scale of the data enabled us to specifically analyze initiation sites and show that these sites have confined locations that are consistently used among individual cells. Further, we find that initiation sites are activated in a similar, albeit not fixed, order across cells. Taken together, our results suggest that replication timing variability is constrained both spatially and temporally, and that the degree of variation is consistent across human cell lines.

Temporal order of DNA replication in the H-2 major histocompatibility complex of the mouse

1992 ◽  
Vol 12 (11) ◽  
pp. 5174-5188
Author(s):  
E G Spack ◽  
E D Lewis ◽  
B Paradowski ◽  
R T Schimke ◽  
P P Jones
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Temporal Order ◽  
Chromosomal Region ◽  
S Phase ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
T Lymphoma Cells ◽  
Initiation Of Dna Replication ◽  
T Lymphoma

As an approach to mapping replicons in an extended chromosomal region, the temporal order of DNA replication was analyzed in the murine major histocompatibility gene complex (MHC). Replicating DNA from T-lymphoma and myelomonocyte cell lines was density labeled with bromodeoxyuridine and extracted from cells which had been fractionated into different stages of S phase by centrifugal elutriation. The replicating DNA from each fraction of S phase was separated from nonreplicating DNA on density gradients, blotted, and hybridized with 34 specific MHC probes. The earliest replication occurred in the vicinity of transcribed genes K, HAM1 and HAM2, RD, B144, D, L, T18, and T3. The temporal order of replication of groups of DNA segments suggests the location of five or six replicons within the H-2 complex, some of which appear to be either unidirectional or markedly asymmetric. The rates of replication through each of these apparent replicons appear to be similar. The TL region of the S49.1 T-lymphoma cells, which contains at least three transcribed genes, replicates earlier than the inactive TL region of WEHI-3 myelomonocytic cells. These results provide further evidence of a relationship between transcription and the initiation of DNA replication in mammalian cells. The mouse MHC examined in this study is the largest chromosomal region (> 2,000 kb) measured for timing of replication to date.

Goosecoid suppresses cell growth and enhances neuronal differentiation of PC12 cells

2000 ◽  
Vol 113 (15) ◽  
pp. 2705-2713
Author(s):  
K. Sawada ◽  
Y. Konishi ◽  
M. Tominaga ◽  
Y. Watanabe ◽  
J. Hirano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neurite Outgrowth ◽  
Neuronal Differentiation ◽  
Pc12 Cells ◽  
Mammalian Cells ◽  
Homeobox Gene ◽  
S Phase ◽  
Bhlh Transcription Factor ◽  
Spemann Organizer

In all vertebrate species, the homeobox gene goosecoid serves as a marker of the Spemann organizer tissue. One function of the organizer is the induction of neural tissue. To investigate the role of goosecoid in neuronal differentiation of mammalian cells, we have introduced goosecoid into PC12 cells. Expression of goosecoid resulted in reduced cell proliferation and enhanced neurite outgrowth in response to NGF. Expression of goosecoid led to a decrease in the percentage of S-phase cells and to upregulation of the expression of the neuron-specific markers MAP-1b and neurofilament-L. Analysis of goosecoid mutants revealed that these effects were independent of either DNA binding or homodimerization of Goosecoid. Coexpression of the N-terminal portion of the ets transcription factor PU.1, a protein that can bind to Goosecoid, repressed neurite outgrowth and rescued the proliferation of PC12 cultures. In contrast, expression of the bHLH transcription factor HES-1 repressed goosecoid-mediated neurite outgrowth without changing the proportion of S-phase cells. These results suggest that goosecoid is involved in neuronal differentiation in two ways, by slowing the cell cycle and stimulating neurite outgrowth, and that these two events are separately regulated.

DNA precursor pools and ribonucleotide reductase activity: distribution between the nucleus and cytoplasm of mammalian cells

1985 ◽  
Vol 5 (12) ◽  
pp. 3443-3450
Author(s):  
J M Leeds ◽  
M B Slabaugh ◽  
C K Mathews
Keyword(s):  
Cell Cycle ◽  
Ribonucleotide Reductase ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Cell Activity ◽  
S Phase ◽  
Whole Cell ◽  
Ribonucleotide Reductase Activity

Nuclear and whole-cell deoxynucleoside triphosphate (dNTP) pools were measured in HeLa cells at different densities and throughout the cell cycle of synchronized CHO cells. Nuclei were prepared by brief detergent (Nonidet P-40) treatment of subconfluent monolayers, a procedure that solubilizes plasma membranes but leaves nuclei intact and attached to the plastic substratum. Electron microscopic examination of monolayers treated with Nonidet P-40 revealed protruding nuclei surrounded by cytoskeletal remnants. Control experiments showed that nuclear dNTP pool sizes were stable during the time required for isolation, suggesting that redistribution of nucleotides during the isolation procedure was minimal. Examination of HeLa whole-cell and nuclear dNTP levels revealed that the nuclear proportion of each dNTP was distinct and remained constant as cell density increased. In synchronized CHO cells, all four dNTP whole-cell pools increased during S phase, with the dCTP pool size increasing most dramatically. The nuclear dCTP pool did not increase as much as the whole-cell dCTP pool during S phase, lowering the relative nuclear dCTP pool. Although the whole-cell dNTP pools decreased after 30 h of isoleucine deprivation, nuclear pools did not decrease proportionately. In summary, nuclear dNTP pools in synchronized CHO cells maintained a relatively constant concentration throughout the cell cycle in the face of larger fluctuations in whole-cell dNTP pools. Ribonucleotide reductase activity was measured in CHO cells throughout the cell cycle, and although there was a 10-fold increase in whole-cell activity during S phase, we detected no reductase in nuclear preparations at any point in the cell cycle.

Cell cycle variations of dinucleoside polyphosphates in synchronized cultures of mammalian cells

1987 ◽  
Vol 7 (7) ◽  
pp. 2444-2450
Author(s):  
G Orfanoudakis ◽  
M Baltzinger ◽  
D Meyer ◽  
N Befort ◽  
J P Ebel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
S Phase ◽  
Serum Deprivation ◽  
Specific Cell ◽  
Culture Cells ◽  
Mouse Embryo Fibroblasts ◽  
Atp Pool ◽  
Dinucleoside Polyphosphates ◽  
Synchronized Cultures

Zajdela hepatoma culture cells (ZHC) and mouse embryo fibroblasts (Swiss 3T3) were synchronized in G1 or S phase by serum deprivation and aphidicolin treatment, respectively, to study the variations in adenylyl nucleotide (Ap4X) pool size during the progress of the cell cycle. Only minor variations, which never exceeded a factor of 2, were observed when the Ap4X concentrations were expressed on a cellular basis. The variations were found to be strictly parallel to the ATP variations. Upon release from an aphidicolin block, the minor variations of Ap4X followed DNA synthesis and preceded cytokinesis. When the nucleotide content was compared with the amount of proteins, the faint specific cell cycle changes were almost completely damped when the cells were synchronized by serum deprivation, but remained practically unchanged in the case of aphidicolin synchronization. These results suggest that the observed variations could reflect the accumulation of some nucleotides before cell division. It is not clear yet whether the variation in Ap4X concentration is significant by itself or is simply a phenomenon resulting from changes in the ATP pool.

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