A journey through the microscopic ages of DNA replication

Reinhart, Marius; Cardoso, M. Cristina

doi:10.1007/s00709-016-1058-8

Cited by 7 publications

(4 citation statements)

References 174 publications

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“…Labeling cells with modified nucleotides revealed that the replicon clusters observed on DNA fibers become visible as the before-mentioned replication foci in interphase nuclei ( 15 ). With higher optical resolution levels, the number of replication foci measured in cells increased and each replication nanofocus in somatic mammalian cells was shown to be equivalent to a replicon unit ( 28 , 29 ). Besides loop structures, chromatin signatures and the associated changes in chromatin structure and accessibility, influence licensing and activation of origins of replication and, thus, replication timing programs in mammalian cells ( 30 ).…”

Section: Introductionmentioning

confidence: 99%

Developmental differences in genome replication program and origin activation

Rausch

Weber

Prorok

et al. 2020

Nucleic Acids Research

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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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Section: Introductionmentioning

confidence: 99%

Developmental differences in genome replication program and origin activation

Rausch

Weber

Prorok

et al. 2020

Nucleic Acids Research

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“…1A), for review see [9,10,12,13]. Microscopic studies have essentially contributed to the current state of knowledge on replication [16]. For instance, early studies on stretched DNA fibers, prepared from nuclei after pulse-replication labeling, revealed that several adjacent replicons are synchronously activated resulting in replication domains (RDs) with a length of ~0.5 -1Mb [17] (Fig.…”

Section: Dna Replication a Key Process Of Nuclear Functionmentioning

confidence: 99%

Perspective Article: Space-time dynamics of genome replication studied with super-resolved microscopy

Gelleri,

Sterr,

Strickfaden

et al. 2024

Postepy Biochem

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Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.

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“…Advanced optical microscopy techniques have been used to investigate the replisome localization and dynamics also in eukaryotes ( 11 , 12 ). These studies have underlined how technical developments in optical microscopy methods beyond the Abbe (diffraction) limit have changed our views on the intranuclear organization of DNA replication.…”

Section: Introductionmentioning

confidence: 99%

Snapshots of archaeal DNA replication and repair in living cells using super-resolution imaging

Delpech

Collien

Mahou

et al. 2018

Nucleic Acids Research

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Using the haloarchaeon Haloferax volcanii as a model, we developed nascent DNA labeling and the functional GFP-labeled single-stranded binding protein RPA2 as novel tools to gain new insight into DNA replication and repair in live haloarchaeal cells. Our quantitative fluorescence microscopy data revealed that RPA2 forms distinct replication structures that dynamically responded to replication stress and DNA damaging agents. The number of the RPA2 foci per cell followed a probabilistic Poisson distribution, implying hitherto unnoticed stochastic cell-to-cell variation in haloarchaeal DNA replication and repair processes. The size range of haloarchaeal replication structures is very similar to those observed earlier in eukaryotic cells. The improved lateral resolution of 3D-SIM fluorescence microscopy allowed proposing that inhibition of DNA synthesis results in localized replication foci clustering and facilitated observation of RPA2 complexes brought about by chemical agents creating DNA double-strand breaks. Altogether our in vivo observations are compatible with earlier in vitro studies on archaeal single-stranded DNA binding proteins. Our work thus underlines the great potential of live cell imaging for unraveling the dynamic nature of transient molecular interactions that underpin fundamental molecular processes in the Third domain of life.

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A journey through the microscopic ages of DNA replication

Cited by 7 publications

References 174 publications

Developmental differences in genome replication program and origin activation

Developmental differences in genome replication program and origin activation

Perspective Article: Space-time dynamics of genome replication studied with super-resolved microscopy

Snapshots of archaeal DNA replication and repair in living cells using super-resolution imaging

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