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.

scholarly journals Caught in the act: structural dynamics of replication origin activation and fork progression

2020 ◽  
Vol 48 (3) ◽  
pp. 1057-1066 ◽  
Author(s):  
Jacob S. Lewis ◽  
Alessandro Costa
Keyword(s):  
Dna Replication ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Single Particle ◽  
Replication Origin ◽  
Origin Activation ◽  
Recent Advances ◽  
Eukaryotic Dna ◽  
New Challenges

This review discusses recent advances in single-particle cryo-EM and single-molecule approaches used to visualise eukaryotic DNA replication reactions reconstituted in vitro. We comment on the new challenges facing structural biologists, as they turn to describing the dynamic cascade of events that lead to replication origin activation and fork progression.

scholarly journals Chromosomal Mcm2-7 distribution and the genome replication program in species from yeast to humans

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009714
Author(s):  
Eric J. Foss ◽  
Smitha Sripathy ◽  
Tonibelle Gatbonton-Schwager ◽  
Hyunchang Kwak ◽  
Adam H. Thiesen ◽  
...  
Keyword(s):  
Binding Sites ◽  
Budding Yeast ◽  
Replication Timing ◽  
S Phase ◽  
Genome Replication ◽  
Sequence Specificity ◽  
Chromosomal Distribution ◽  
Chromosomal Replication ◽  
Inactive X Chromosome ◽  
Spatio Temporal

The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we mapped the binding sites of the Mcm2-7 helicase complex (MCM) in budding yeast, fission yeast, mouse and humans. We observed similar individual MCM double-hexamer (DH) footprints across the species, but notable differences in their distribution: Footprints in budding yeast were more sharply focused compared to the other three organisms, consistent with the relative sequence specificity of replication origins in S. cerevisiae. Nonetheless, with some clear exceptions, most notably the inactive X-chromosome, much of the fluctuation in replication timing along the chromosomes in all four organisms reflected uneven chromosomal distribution of pre-replication complexes.

scholarly journals Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins

1999 ◽  
Vol 112 (12) ◽  
pp. 2011-2018 ◽  
Author(s):  
A. Rowles ◽  
S. Tada ◽  
J.J. Blow
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Origin ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Replication Origins ◽  
Free System ◽  
Essential Function ◽  
Initiation Of Dna Replication ◽  
Origin Recognition

During late mitosis and early G1, a series of proteins are assembled onto replication origins that results in them becoming ‘licensed’ for replication in the subsequent S phase. In Xenopus this first involves the assembly onto chromatin of the Xenopus origin recognition complex XORC, and then XCdc6, and finally the RLF-M component of the replication licensing system. In this paper we examine changes in the way that XORC associates with chromatin in the Xenopus cell-free system as origins become licensed. Restricting the quantity of XORC on chromatin reduced the extent of replication as expected if a single molecule of XORC is sufficient to specify a single replication origin. During metaphase, XOrc1 associated only weakly with chromatin. In early interphase, XOrc1 formed a strong complex with chromatin, as evidenced by its resistance to elution by 200 mM salt, and this state persisted when XCdc6 was assembled onto the chromatin. As a consequence of origins becoming licensed the association of XOrc1 and XCdc6 with chromatin was destabilised, and XOrc1 became susceptible to removal from chromatin by exposure to either high salt or high Cdk levels. At this stage the essential function for XORC and XCdc6 in DNA replication had already been fulfilled. Since high Cdk levels are required for the initiation of DNA replication, this ‘licensing-dependent origin inactivation’ may contribute to mechanisms that prevent re-licensing of replication origins once S phase has started.

Cell cycle-dependent calcium oscillations in mouse embryonic stem cells

2007 ◽  
Vol 292 (4) ◽  
pp. C1510-C1518 ◽  
Author(s):  
Nidhi Kapur ◽  
Gregory A. Mignery ◽  
Kathrin Banach
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Es Cells ◽  
Embryonic Stem ◽  
S Phase ◽  
Calcium Oscillations ◽  
Confocal Imaging ◽  
Cycle Progression ◽  
Intracellular Ca ◽  
Mes Cells

During cell cycle progression, somatic cells exhibit different patterns of intracellular Ca2+signals during the G0phase, the transition from G1to S, and from G2to M. Because pluripotent embryonic stem (ES) cells progress through cell cycle without the gap phases G1and G2, we aimed to determine whether mouse ES (mES) cells still exhibit characteristic changes of intracellular Ca2+concentration during cell cycle progression. With confocal imaging of the Ca2+-sensitive dye fluo-4 AM, we identified that undifferentiated mES cells exhibit spontaneous Ca2+oscillations. In control cultures where 50.4% of the cells reside in the S phase of the cell cycle, oscillations appeared in 36% of the cells within a colony. Oscillations were not initiated by Ca2+influx but depended on inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+release and the refilling of intracellular stores by a store-operated Ca2+influx (SOC) mechanism. Using cell cycle synchronization, we determined that Ca2+oscillations were confined to the G1/S phase (∼70% oscillating cells vs. G2/M with ∼15% oscillating cells) of the cell cycle. ATP induced Ca2+oscillations, and activation of SOC could be induced in G1/S and G2/M synchronized cells. Intracellular Ca2+stores were not depleted, and all three IP3receptor isoforms were present throughout the cell cycle. Cell cycle analysis after EGTA, BAPTA-AM, 2-aminoethoxydiphenyl borate, thapsigargin, or U-73122 treatment emphasized that IP3-mediated Ca2+release is necessary for cell cycle progression through G1/S. Because the IP3receptor sensitizer thimerosal induced Ca2+oscillations only in G1/S, we propose that changes in IP3receptor sensitivity or basal levels of IP3could be the basis for the G1/S-confined Ca2+oscillations.

scholarly journals On the Interplay of the DNA Replication Program and the Intra-S Phase Checkpoint Pathway

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 94 ◽  
Author(s):  
Diletta Ciardo ◽  
Arach Goldar ◽  
Kathrin Marheineke
Keyword(s):  
Dna Replication ◽  
Genome Stability ◽  
Current Knowledge ◽  
Theoretical Models ◽  
S Phase ◽  
Origin Firing ◽  
Checkpoint Pathway ◽  
M Phase ◽  
Spatio Temporal ◽  
S Phase Checkpoint

DNA replication in eukaryotes is achieved by the activation of multiple replication origins which needs to be precisely coordinated in space and time. This spatio-temporal replication program is regulated by many factors to maintain genome stability, which is frequently threatened through stresses of exogenous or endogenous origin. Intra-S phase checkpoints monitor the integrity of DNA synthesis and are activated when replication forks are stalled. Their activation leads to the stabilization of forks, to the delay of the replication program by the inhibition of late firing origins, and the delay of G2/M phase entry. In some cell cycles during early development these mechanisms are less efficient in order to allow rapid cell divisions. In this article, we will review our current knowledge of how the intra-S phase checkpoint regulates the replication program in budding yeast and metazoan models, including early embryos with rapid S phases. We sum up current models on how the checkpoint can inhibit origin firing in some genomic regions, but allow dormant origin activation in other regions. Finally, we discuss how numerical and theoretical models can be used to connect the multiple different actors into a global process and to extract general rules.

scholarly journals High Resolution Repli-Seq defines the temporal choreography of initiation, elongation and termination of replication in mammalian cells

2019 ◽  
Author(s):  
Peiyao A. Zhao ◽  
Takayo Sasaki ◽  
David M. Gilbert
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Replication Timing ◽  
Cell Types ◽  
S Phase ◽  
List Type ◽  
Cell Type ◽  
Histone Marks ◽  
Cell Type Specific

ABSTRACTDNA replication in mammalian cells occurs in a defined temporal order during S phase, known as the replication timing (RT) programme. RT is developmentally regulated and correlated with chromatin conformation and local transcriptional potential. Here we present RT profiles of unprecedented temporal resolution in two human embryonic stem cell lines, human colon carcinoma line HCT116 as well as F1 subspecies hybrid mouse embryonic stem cells and their neural progenitor derivatives. Strong enrichment of nascent DNA in fine temporal windows reveals a remarkable degree of cell to cell conservation in replication timing and patterns of replication genome-wide. We identify 5 patterns of replication in all cell types, consistent with varying degrees of initiation efficiency. Zones of replication initiation were found throughout S phase and resolved to ~50kb precision. Temporal transition regions were resolved into segments of uni-directional replication punctuated with small zones of inefficient initiation. Small and large valleys of convergent replication were consistent with either termination or broadly distributed initiation, respectively. RT correlated with chromatin compartment across all cell types but correlations of initiation time to chromatin domain boundaries and histone marks were cell type specific. Haplotype phasing revealed previously unappreciated regions of allele-specific and alleleindependent asynchronous replication. Allele-independent asynchrony was associated with large transcribed genes that resemble common fragile sites. Altogether, these data reveal a remarkably deterministic temporal choreography of DNA replication in mammalian cells.Highly homogeneous replication landscape between cells in a populationInitiation zones resolved within constant timing and timing transition regionsActive histone marks enriched within early initiation zones while enrichment of repressive marks is cell type specific.Transcribed long genes replicate asynchronously.

scholarly journals A non-transcriptional function of Yap orchestrates the DNA replication program

2021 ◽  
Author(s):  
Rodrigo Meléndez García ◽  
Olivier Haccard ◽  
Albert Chesneau ◽  
Hemalatha Narassimprakash ◽  
Jerome E Roger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Replication ◽  
Embryonic Stem ◽  
Replication Timing ◽  
S Phase ◽  
Hippo Signaling ◽  
Downstream Effector ◽  
Egg Extracts ◽  
Replication Foci ◽  
Eukaryotic Organisms

In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signaling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and that Yap depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. Furthermore, we identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that both Yap and Rif1 depletion trigger an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as breaks to control the DNA replication program in early embryos and post-embryonic stem cells.

scholarly journals Genome-Wide Mapping of Human DNA Replication by Optical Replication Mapping Supports a Stochastic Model of Eukaryotic Replication

2020 ◽  
Author(s):  
Weitao Wang ◽  
Kyle Klein ◽  
Karel Proesmans ◽  
Hongbo Yang ◽  
Claire Marchal ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Qualitative Difference ◽  
S Phase ◽  
Human Cells ◽  
Replication Initiation ◽  
Early Initiation ◽  
Firing Time ◽  
Genome Wide ◽  
Low Efficiency

AbstractDNA replication is regulated by the location and timing of replication initiation. Therefore, much effort has been invested in identifying and analyzing the sites of human replication initiation. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation site utilization in metazoans has made mapping the location and timing of replication initiation in human cells difficult. A potential solution to the problem of human replication mapping is single-molecule analysis. However, current approaches do not provide the throughput required for genome-wide experiments. To address this challenge, we have developed Optical Replication Mapping (ORM), a high-throughput single-molecule approach to map newly replicated DNA, and used it to map early initiation events in human cells. The single-molecule nature of our data, and a total of more than 2000-fold coverage of the human genome on 27 million fibers averaging ~300 kb in length, allow us to identify initiation sites and their firing probability with high confidence. In particular, for the first time, we are able to measure genome-wide the absolute efficiency of human replication initiation. We find that the distribution of human replication initiation is consistent with inefficient, stochastic initiation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. In particular, we find sites of human replication initiation are not confined to well-defined replication origins but are instead distributed across broad initiation zones consisting of many initiation sites. Furthermore, we find no correlation of initiation events between neighboring initiation zones. Although most early initiation events occur in early-replicating regions of the genome, a significant number occur in late-replicating regions. The fact that initiation sites in typically late-replicating regions have some probability of firing in early S phase suggests that the major difference between initiation events in early and late replicating regions is their intrinsic probability of firing, as opposed to a qualitative difference in their firing-time distributions. Moreover, modeling of replication kinetics demonstrates that measuring the efficiency of initiation-zone firing in early S phase suffices to predict the average firing time of such initiation zones throughout S phase, further suggesting that the differences between the firing times of early and late initiation zones are quantitative, rather than qualitative. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

scholarly journals The ATR-Activation Domain of TopBP1 Is Required for the Suppression of Origin Firing during the S Phase

2018 ◽  
Vol 19 (8) ◽  
pp. 2376 ◽  
Author(s):  
Miiko Sokka ◽  
Dennis Koalick ◽  
Peter Hemmerich ◽  
Juhani Syväoja ◽  
Helmut Pospiech
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Activation Domain ◽  
Origin Firing ◽  
Dna Topoisomerase ◽  
Origin Activation ◽  
Topoisomerase 2 ◽  
Initiation Of Dna Replication ◽  
Two Phases ◽  
Atr Activation

The mammalian DNA replication program is controlled at two phases, the licensing of potential origins of DNA replication in early gap 1 (G1), and the selective firing of a subset of licenced origins in the synthesis (S) phase. Upon entry into the S phase, serine/threonine-protein kinase ATR (ATR) is required for successful completion of the DNA replication program by limiting unnecessary dormant origin activation. Equally important is its activator, DNA topoisomerase 2-binding protein 1 (TopBP1), which is also required for the initiation of DNA replication after a rise in S-phase kinase levels. However, it is unknown how the ATR activation domain of TopBP1 affects DNA replication dynamics. Using human cells conditionally expressing a TopBP1 mutant deficient for ATR activation, we show that functional TopBP1 is required in suppressing local dormant origin activation. Our results demonstrate a regulatory role for TopBP1 in the local balancing of replication fork firing within the S phase.

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