A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells

Ahuja, Akshay K.; Jodkowska, Karolina; Teloni, Federico; Bizard, Anna H.; Zellweger, Ralph; Herrador, Raquel; Ortega, Sagrario; Hickson, Ian D.; Altmeyer, Matthias; Méndez, Juan Pablo; Lopes, Massimo

doi:10.1038/ncomms10660

Cited by 166 publications

(172 citation statements)

References 53 publications

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“…Using time-lapse imaging, we analyzed the whole cell cycle of 46 single haploid and diploid ESCs, respectively (Figures 1G, 1H, S1B, and S1C; Movies S1 and S2). Interestingly, while the cell-cycle lengths of our diploid ESCs were consistent with previous reports (Figures 1I and S1D; Ahuja et al., 2016, Coronado et al., 2013, Re et al., 2014, Roccio et al., 2013), haploid ESCs exhibited a significantly prolonged cell cycle (Figures 1H, 1I, S1C, and S1D). Statistical analysis showed that the duration of the G1 phase was almost the same in both haploid and diploid ESCs, but the total length of the S and G2/M phases was about 5 hr longer in haploid ESCs than in diploid cells (Figures 1I, 1J, S1D, and S1E), consistent with a marginal although not statistically significant increase of the S-G2/M proportion in haploid cells (Figures 1J and S1E).…”

Section: Resultssupporting

confidence: 93%

Single-Cell Dynamic Analysis of Mitosis in Haploid Embryonic Stem Cells Shows the Prolonged Metaphase and Its Association with Self-diploidization

Guo

Huang

et al. 2017

Stem Cell Reports

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SummaryThe recent establishment of mammalian haploid embryonic stem cells (ESCs) provides new possibilities for genetic screening and for understanding genome evolution and function. However, the dynamics of mitosis in haploid ESCs is still unclear. Here, we report that the duration of mitosis in haploid ESCs, especially the metaphase, is significantly longer than that in diploid ESCs. Delaying mitosis by chemicals increased self-diploidization of haploid ESCs, while shortening mitosis stabilized haploid ESCs. Taken together, our study suggests that the delayed mitosis of haploid ESCs is associated with self-diploidization.

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Section: Resultssupporting

confidence: 93%

Single-Cell Dynamic Analysis of Mitosis in Haploid Embryonic Stem Cells Shows the Prolonged Metaphase and Its Association with Self-diploidization

Guo

Huang

et al. 2017

Stem Cell Reports

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“…We did not detect changes in the levels of ss C-rich linear telomeric DNA following replication stress, demonstrating that resolution of replication intermediates specifically enriches for C-rich telomeric DNA in circular conformation. We suggest that hESCs accumulate higher levels of replication stress compared to differentiated cells, which is consistent with the demonstration that replication intermediates are frequently observed in ESC, but lost after differentiation 57 .…”

Section: Discussionsupporting

confidence: 91%

A balance between elongation and trimming regulates telomere stability in stem cells

Rivera

Haggblom

Cosconati

et al. 2016

Nat Struct Mol Biol

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Telomere length maintenance ensures self-renewal of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), however the mechanisms governing telomere length homeostasis in these cell types are unclear. Here, we report that telomere length is determined by the balance between telomere elongation mediated by telomerase and telomere trimming, controlled by the homologous recombination proteins XRCC3 and Nbs1 that generate single-stranded C-rich telomeric DNA and double-stranded telomeric circular DNA (T-circles), respectively. We found that reprogramming of differentiated cells induces T-circle and single stranded C-rich telomeric DNA accumulation, indicating the activation of telomere trimming pathways that compensate telomerase dependent telomere elongation in hiPSCs. Excessive telomere elongation compromises telomere stability and promotes the formation of partially single-stranded telomeric DNA circles (C-circles) in hESCs, suggesting heightened sensitivity of stem cells to replication stress at overly long telomeres. Thus, tight control of telomere length homeostasis is essential to maintain telomere stability in hESCs.

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“…Because origin licensing occurs in G1, daughters have more opportunity to license origins throughout the genome compared with mother cells, which have a remarkably short G1. In embryonic stem cells, which also exhibit rapid cycling with short G1, reduced licensing of replication origins creates constitutive replication stress (27). A shift in origin firing toward repetitive sequences likely also occurs during cellular aging in humans, where origins are determined by chromatin accessibility (28,29), and heterochromatic repetitive sequences open up with age (3, 4).…”

Section: Discussionmentioning

confidence: 99%

SIR2 suppresses replication gaps and genome instability by balancing replication between repetitive and unique sequences

Foss

Lao

Dalrymple

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Replication gaps that persist into mitosis likely represent important threats to genome stability, but experimental identification of these gaps has proved challenging. We have developed a technique that allows us to explore the dynamics by which genome replication is completed before mitosis. Using this approach, we demonstrate that excessive allocation of replication resources to origins within repetitive regions, induced by SIR2 deletion, leads to persistent replication gaps and genome instability. Conversely, the weakening of replication origins in repetitive regions suppresses these gaps. Given known age-and cancer-associated changes in chromatin accessibility at repetitive sequences, we suggest that replication gaps resulting from misallocation of replication resources underlie age-and disease-associated genome instability.SIR2 | DNA replication | repetitive sequences | replication gaps | ribosomal DNA S taggered initiation of DNA replication, which is common across eukaryotes from fungi to humans, means that, at any given time, in S phase, only a fraction of replication origins is activated. In recent years, a model has emerged to explain this pattern of DNA replication (1, 2). This model assumes that the pool of initiation factors required to fire licensed origins in S phase is limited, and therefore sufficient to fire only a subset of licensed origins at any given time. Licensed origins differ in their ability to recruit factors required for firing, so origins with higher affinity or accessibility fire earlier than those with lower affinity or accessibility. After activating the initial set of origins, firing factors are released, enabling the next set of origins to fire. This results in successive waves of origin activation. Genome replication eventually finishes when areas with the least accessible origins are replicated.In healthy human cells, the least accessible genomic regions consist of repetitive DNA, which represents about half of the genome and tends to be compacted into heterochromatin. However, recent studies suggest widespread opening of hetrochromatin during carcinogenesis and aging (3-5). Such reorganization of chromatin would expose a new suite of origins within repetitive DNA that could potentially compete initiation factors away from unique portions of the genome, thereby disrupting the normal genome-wide hierarchy of replication timing. Increased origin activity within repetitive DNA could thus compromise replication elsewhere in the genome. Given the limited pool of initiation factors, we propose that an increase in density of active origins within repetitive regions could result in a decreased density of such origins in unique regions of the genome, which, when combined with the stochastic nature of origin firing, may occasionally result in replication gaps, i.e., unique regions of the genome that are unable to complete replication before mitosis. This so-called "Random Replication Gap Problem" (RRGP) is the subject of long-standing speculation, but such gaps have never been experim...

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A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells

Cited by 166 publications

References 53 publications

Single-Cell Dynamic Analysis of Mitosis in Haploid Embryonic Stem Cells Shows the Prolonged Metaphase and Its Association with Self-diploidization

Single-Cell Dynamic Analysis of Mitosis in Haploid Embryonic Stem Cells Shows the Prolonged Metaphase and Its Association with Self-diploidization

A balance between elongation and trimming regulates telomere stability in stem cells

SIR2 suppresses replication gaps and genome instability by balancing replication between repetitive and unique sequences

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