scholarly journals The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells

2002 ◽  
Vol 115 (21) ◽  
pp. 4037-4051 ◽  
Author(s):  
D. S. Dimitrova
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Temporal Organization ◽  
Replication Sites ◽  
Spatio Temporal

scholarly journals Stochasticity of replication fork speed plays a key role in the dynamics of DNA replication

2019 ◽  
Author(s):  
Razie Yousefi ◽  
Maga Rowicka
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Computational Models ◽  
Replication Timing ◽  
Temporal Organization ◽  
Yeast Cells ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Timely Completion ◽  
Spatio Temporal

AbstractEukaryotic DNA replication is elaborately orchestrated to duplicate the genome timely and faithfully. Replication initiates at multiple origins from which replication forks emanate and travel bi-directionally. The complex spatio-temporal regulation of DNA replication remains incompletely understood. To study it, computational models of DNA replication have been developed in S. cerevisiae. However, in spite of the experimental evidence of replication speed stochasticity, all models assumed that replication fork speed is constant or varies only with genomic coordinates. Here, we present the first model of DNA replication assuming stochastic speed of the replication fork. Utilizing data from both wild-type and hydroxyurea-treated yeast cells, we show that our model is more accurate than models assuming constant fork speed and reconstructs dynamics of DNA replication faithfully starting both from population-wide data and data reflecting fork movement in individual cells. Completion of replication in a timely manner is a challenge due to its stochasticity; we propose an empirically derived modification to replication speed based on the distance to the approaching fork, which promotes timely completion of replication. In summary, our work discovers a key role that stochasticity of the fork speed plays in the dynamics of DNA replication. We show that without including stochasticity of fork speed it is not possible to accurately reconstruct movement of individual replication forks, measured by DNA combing.Author summaryDNA replication in eukaryotes starts from multiple sites termed replication origins. Replication timing at individual sites is stochastic, but reproducible population-wide. Complex and not yet completely understood mechanisms ensure that genome is replicated exactly once and that replication is finished in time. This complex spatio-temporal organization of DNA replication makes computational modeling a useful tool to study replication mechanisms. For simplicity, all previous models assumed constant replication fork speed. Here, we show that such models are incapable of accurately reconstructing distances travelled by individual replication forks. Therefore, we propose a model with a stochastic replication fork speed. We show that such model reproduces faithfully distances travelled by individual replication forks. Moreover, our model is simpler than previous model and thus avoids over-learning (fitting noise). We also discover how replication speed may be attuned to timely complete replication. We propose that fork speed exponentially increases with diminishing distance to the approaching fork, which we show promotes timely completion of replication. Such speed up can be e.g. explained by a synergy effect of chromatin unwinding by both forks. Our model can be used to simulate phenomena beyond replication, e.g. DNA double-strand breaks resulting from broken replication forks.

scholarly journals Nuclear IE2 Structures Are Related to Viral DNA Replication Sites during Baculovirus Infection

2002 ◽  
Vol 76 (10) ◽  
pp. 5198-5207 ◽  
Author(s):  
Daniela Mainz ◽  
Ilja Quadt ◽  
Dagmar Knebel-Mörsdorf
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Confocal Imaging ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Functional Sites ◽  
Baculovirus Infection ◽  
Replication Sites ◽  
Early Replication ◽  
Nuclear Polyhedrosis

ABSTRACT The ie2 gene of Autographa californica multicapsid nuclear polyhedrosis virus is 1 of the 10 baculovirus genes that have been identified as factors involved in viral DNA replication. IE2 is detectable in the nucleus as one of the major early-expressed proteins and exhibits a dynamic localization pattern during the infection cycle (D. Murges, I. Quadt, J. Schröer, and D. Knebel-Mörsdorf, Exp. Cell Res. 264:219-232, 2001). Here, we investigated whether IE2 localized to regions of viral DNA replication. After viral DNA was labeled with bromodeoxyuridine (BrdU), confocal imaging indicated that defined IE2 domains colocalized with viral DNA replication centers as soon as viral DNA replication was detectable. In addition, a subpopulation of IE2 structures colocalized with two further virus-encoded replication factors, late expression factor 3 (LEF-3) and the DNA binding protein (DBP). While DBP and LEF-3 structures always colocalized and enlarged simultaneously with viral DNA replication sites, only those IE2 structures that colocalized with replication sites also colocalized with DBP. Replication and transcription of DNA viruses in association with promyelocytic leukemia protein (PML) oncogenic domains have been observed. By confocal imaging we demonstrated that the human PML colocalized with IE2. Triple staining revealed PML/IE2 domains in the vicinity of viral DNA replication centers, while IE2 alone colocalized with early replication sites, demonstrating that PML structures do not form common domains with viral DNA replication centers. Thus, we conclude that IE2 colocalizes alternately with PML and the sites of viral DNA replication. Small ubiquitin-like modifier SUMO-1 has been implicated in the nuclear distribution of PML. Similar to what was found for mammalian cells, small ubiquitin-like modifiers were recruited to PML domains in infected insect cells, which suggests that IE2 and PML colocalize in conserved cellular domains. In summary, our results support a model for IE2 as part of various functional sites in the nucleus that are connected with viral DNA replication.

scholarly journals Temporal organization of replication in DNA fibers of mammalian cells.

1979 ◽  
Vol 81 (3) ◽  
pp. 692-697 ◽  
Author(s):  
B R Jasny ◽  
I Tamm
Keyword(s):  
Dna Replication ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Temporal Organization ◽  
Control Mechanisms ◽  
Mammalian Cell Lines ◽  
Quantitative Estimates ◽  
Coordinate Control ◽  
Mouse Cells ◽  
L929 Mouse

The extent of coordinate control over the multiple initiation events in DNA replication has been investigated in three mammalian cell lines by DNA fiber autoradiography. Quantitative estimates have been obtained of the degree of synchrony among initiations occurring on stretches of DNA. Synchrony decreases markedly with increasing distance between initiation sites in MDBK (bovine) and L929 (mouse) cells, but only slightly in muntjac cells. Possible control mechanisms for the initiation process are discussed.

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.

DNA Replication Sites within Nuclei of Mammalian Cells

Nature ◽  
1973 ◽  
Vol 241 (5384) ◽  
pp. 32-36 ◽  
Author(s):  
JOEL A. HUBERMAN ◽  
ALICE TSAI ◽  
ROBERT A. DEICH
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Replication Sites

scholarly journals Developmental differences in genome replication program and origin activation

2020 ◽  
Vol 48 (22) ◽  
pp. 12751-12777
Author(s):  
Cathia Rausch ◽  
Patrick Weber ◽  
Paulina Prorok ◽  
David Hörl ◽  
Andreas Maiser ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Embryonic Stem ◽  
S Phase ◽  
Developmental Differences ◽  
Centromeric Heterochromatin ◽  
Genome Replication ◽  
Origin Activation ◽  
Spatio Temporal ◽  
Mes Cells

Abstract To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, we observe mid S-phase replication of (peri)centromeric heterochromatin in mES cells, which switches to late S-phase replication upon differentiation. This replication timing reversal correlates with and depends on an increase in condensation and a decrease in acetylation of chromatin. We further find synchronous duplication of the Y chromosome, marking the end of S-phase, irrespectively of the pluripotency state. Using a combination of single-molecule and super-resolution microscopy, we measure molecular properties of the mES cell replicon, the number of replication foci active in parallel and their spatial clustering. We conclude that each replication nanofocus in mES cells corresponds to an individual replicon, with up to one quarter representing unidirectional forks. Furthermore, with molecular combing and genome-wide origin mapping analyses, we find that mES cells activate twice as many origins spaced at half the distance than somatic cells. Altogether, our results highlight fundamental developmental differences on progression of genome replication and origin activation in pluripotent cells.

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