scholarly journals Mechanisms of genetic instability in a single S-phase following whole genome doubling

2021 ◽  
Author(s):  
Simon Gemble ◽  
Sara Vanessa Bernhard ◽  
Nishit Srivastava ◽  
Rene Wardenaar ◽  
Maddalena Nano ◽  
...  
Keyword(s):  
Dna Replication ◽  
Genetic Instability ◽  
Chromosome Instability ◽  
Rapid Evolution ◽  
Cell Types ◽  
S Phase ◽  
Model Organisms ◽  
Whole Genome ◽  
Mass Scaling ◽  
Protein Mass

Doubling of the full chromosome content -whole genome duplications (WGDs)- is frequently found in human cancers and is responsible for the rapid evolution of genetically unstable karyotypes. It has previously been established that WGDs fuel chromosome instability due to abnormal mitosis owing to the presence of extra centrosomes and extra chromosomes. Tolerance to ploidy changes has been identified in different model organisms and cell types, revealing long term cellular adaptations that accommodate ploidy increase. Importantly, however, the immediate consequences of WGDs as cells become tetraploid are not known. It also remains unknown whether WGD triggers other events leading to genetic instability (GIN), independently of mitosis. In this study, we induced tetraploidy in diploid genetically stable RPE-1 cells and monitored the first interphase. We found that newly born tetraploids undergo high rates of DNA damage during DNA replication. Using DNA combing and single cell sequencing, we show that replication forks are unstable, perturbing DNA replication dynamics and generating under- and over-replicated regions at the end of S-phase. Mechanistically, we found that these defects result from lack of protein mass scaling up at the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can accumulate highly abnormal karyotypes. These findings provide an explanation for the GIN landscape that favors tumorigenesis after tetraploidization.

scholarly journals Deregulation of cyclin E in human cells interferes with prereplication complex assembly

2004 ◽  
Vol 165 (6) ◽  
pp. 789-800 ◽  
Author(s):  
Susanna Ekholm-Reed ◽  
Juan Méndez ◽  
Donato Tedesco ◽  
Anders Zetterberg ◽  
Bruce Stillman ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cell Lines ◽  
Broad Spectrum ◽  
Cyclin E ◽  
Chromosome Instability ◽  
S Phase ◽  
Potential Mechanism ◽  
Minichromosome Maintenance ◽  
Origins Of Replication ◽  
Initiation Of Replication

Deregulation of cyclin E expression has been associated with a broad spectrum of human malignancies. Analysis of DNA replication in cells constitutively expressing cyclin E at levels similar to those observed in a subset of tumor-derived cell lines indicates that initiation of replication and possibly fork movement are severely impaired. Such cells show a specific defect in loading of initiator proteins Mcm4, Mcm7, and to a lesser degree, Mcm2 onto chromatin during telophase and early G1 when Mcm2–7 are normally recruited to license origins of replication. Because minichromosome maintenance complex proteins are thought to function as a heterohexamer, loading of Mcm2-, Mcm4-, and Mcm7-depleted complexes is likely to underlie the S phase defects observed in cyclin E–deregulated cells, consistent with a role for minichromosome maintenance complex proteins in initiation of replication and fork movement. Cyclin E–mediated impairment of DNA replication provides a potential mechanism for chromosome instability observed as a consequence of cyclin E deregulation.

scholarly journals cAMP-mediated Inhibition of DNA Replication and S Phase Progression: Involvement of Rb, p21Cip1, and PCNA

2005 ◽  
Vol 16 (3) ◽  
pp. 1527-1542 ◽  
Author(s):  
Soheil Naderi ◽  
Jean Y.J. Wang ◽  
Tung-Ti Chen ◽  
Kristine B. Gutzkow ◽  
Heidi K. Blomhoff
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
Antitumor Agents ◽  
Cell Types ◽  
S Phase ◽  
3T3 Fibroblasts ◽  
Phase Progression ◽  
Inhibition Of Dna Synthesis

cAMP exerts an antiproliferative effect on a number of cell types including lymphocytes. This effect of cAMP is proposed to be mediated by its ability to inhibit G1/S transition. In this report, we provide evidence for a new mechanism whereby cAMP might inhibit cellular proliferation. We show that elevation of intracellular levels of cAMP inhibits DNA replication and arrests the cells in S phase. The cAMP-induced inhibition of DNA synthesis was associated with the increased binding of p21Cip1to Cdk2-cyclin complexes, inhibition of Cdk2 kinase activity, dephosphorylation of Rb, and dissociation of PCNA from chromatin in S phase cells. The ability of cAMP to inhibit DNA replication and trigger release of PCNA from chromatin required Rb and p21Cip1proteins, since both processes were only marginally affected by increased levels of cAMP in Rb-/-and p21Cip1-/-3T3 fibroblasts. Importantly, the implications of cAMP-induced inhibition of DNA synthesis in cancer treatment was demonstrated by the ability of cAMP to reduce apoptosis induced by S phase–specific cytotoxic drugs. Taken together, these results demonstrate a novel role for cAMP in regulation of DNA synthesis and support a model in which activation of cAMP-dependent signaling protects cells from the effect of S phase–specific antitumor agents.

scholarly journals Progression of meiotic DNA replication is modulated by interchromosomal interaction proteins, negatively by Spo11p and positively by Rec8p

2000 ◽  
Vol 14 (4) ◽  
pp. 493-503 ◽  
Author(s):  
Rita S. Cha ◽  
Beth M. Weiner ◽  
Scott Keeney ◽  
Job Dekker ◽  
N. Kleckner
Keyword(s):  
Dna Replication ◽  
Phase Modulation ◽  
Cell Types ◽  
Strand Break ◽  
S Phase ◽  
Wild Type ◽  
Homolog Pairing ◽  
Defective Mutant ◽  
Phase Progression ◽  
Phase Length

Spo11p is a key mediator of interhomolog interactions during meiosis. Deletion of the SPO11 gene decreases the length of S phase by ∼25%. Rec8p is a key coordinator of meiotic interhomolog and intersister interactions. Deletion of the REC8 gene increases S-phase length, by ∼10% in wild-type and ∼30% in aspo11Δ background. Thus, the progression of DNA replication is modulated by interchromosomal interaction proteins. Thespo11–Y135F DSB (double strand break) catalysis-defective mutant is normal for S-phase modulation and DSB-independent homolog pairing but is defective for later events, formation of DSBs, and synaptonemal complexes. Thus, earlier and later functions of Spo11 are defined. We propose that meiotic S-phase progression is linked directly to development of specific chromosomal features required for meiotic interhomolog interactions and that this feedback process is built upon a more fundamental mechanism, common to all cell types, by which S-phase progression is coupled to development of nascent intersister connections and/or related aspects of chromosome morphogenesis. Roles for Rec8 and/or Spo11 in progression through other stages of meiosis are also revealed.

scholarly journals Cell Cycle Asynchrony Generates DNA Damage at Mitotic Entry in Polyploid Cells

2019 ◽  
Author(s):  
Maddalena Nano ◽  
Anthony Simon ◽  
Carole Pennetier ◽  
Vincent Fraisier ◽  
Veronique Marthiens ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Genetic Instability ◽  
Molecular Mechanisms ◽  
Chromosome Instability ◽  
Time Frame ◽  
Whole Genome ◽  
Mitotic Entry ◽  
Polyploid Cells

AbstractPolyploidy arises from the gain of complete chromosomes sets [1] and is known to promote cancer genome evolution. Recent evidence suggests that a large proportion of human tumours experience whole genome duplications (WGDs), which might favour the generation of highly abnormal karyotypes within a short time frame, rather than in a stepwise manner [2–6]. However, the molecular mechanisms linking whole genome duplication to genetic instability remain poorly understood. Further, possible mechanisms responsible for rapid genome reshuffling have not been described yet. Using repeated cytokinesis failure to induce polyploidization of Drosophila neural stem cells (NSCs, also called neuroblasts - NBs), we investigated the consequences of polyploidy in vivo. Here, we show that polyploid NSCs accumulate high levels of chromosome instability. Surprisingly, we found that DNA damage is generated in a subset of nuclei of polyploid NBs during mitosis, in an asymmetric manner. Importantly, our observations in flies were confirmed in mouse NSCs (mNSCs) after acute cytokinesis inhibition. Interestingly, DNA damage occurs in nuclei that were not ready to enter mitosis but were forced to do so when exposed to the mitotic environment of neighbouring nuclei within the same cell. Additionally, we found that polyploid cells are cell cycle asynchronous and forcing cell cycle synchronization is sufficient to lower the levels of DNA damage generated during mitosis. Overall, this work supports a model in which DNA damage at mitotic entry can generate a mutated genetic landscape that contributes to the onset of genetic instability.

scholarly journals Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences.

1992 ◽  
Vol 116 (5) ◽  
pp. 1095-1110 ◽  
Author(s):  
R T O'Keefe ◽  
S C Henderson ◽  
D L Spector
Keyword(s):  
Dna Replication ◽  
Mammalian Cell ◽  
Dna Sequences ◽  
Satellite Dna ◽  
Cell Types ◽  
S Phase ◽  
Laser Scanning Microscopy ◽  
Alpha Satellite ◽  
Alpha Satellite Dna ◽  
Dense Chromatin

Five distinct patterns of DNA replication have been identified during S-phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus. In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin. As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase. At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin. Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the BrdU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific alpha-satellite DNA sequences indicates that the alpha-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.

scholarly journals Mechanisms of eukaryotic replisome disassembly

2020 ◽  
Vol 48 (3) ◽  
pp. 823-836
Author(s):  
Sara Priego Moreno ◽  
Agnieszka Gambus
Keyword(s):  
Dna Replication ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Dna Lesions ◽  
Model Organisms ◽  
Replicative Helicase ◽  
Complex Process ◽  
Cross Links ◽  
Higher Eukaryotes ◽  
Key Events

DNA replication is a complex process that needs to be executed accurately before cell division in order to maintain genome integrity. DNA replication is divided into three main stages: initiation, elongation and termination. One of the key events during initiation is the assembly of the replicative helicase at origins of replication, and this mechanism has been very well described over the last decades. In the last six years however, researchers have also focused on deciphering the molecular mechanisms underlying the disassembly of the replicative helicase during termination. Similar to replisome assembly, the mechanism of replisome disassembly is strictly regulated and well conserved throughout evolution, although its complexity increases in higher eukaryotes. While budding yeast rely on just one pathway for replisome disassembly in S phase, higher eukaryotes evolved an additional mitotic pathway over and above the default S phase specific pathway. Moreover, replisome disassembly has been recently found to be a key event prior to the repair of certain DNA lesions, such as under-replicated DNA in mitosis and inter-strand cross-links (ICLs) in S phase. Although replisome disassembly in human cells has not been characterised yet, they possess all of the factors involved in these pathways in model organisms, and de-regulation of many of them are known to contribute to tumorigenesis and other pathological conditions.

scholarly journals Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots

2020 ◽  
Author(s):  
Emily E. Wear ◽  
Jawon Song ◽  
Gregory J. Zynda ◽  
Leigh Mickelson-Young ◽  
Chantal LeBlanc ◽  
...  
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Mitotic Cycle ◽  
Genetic Material ◽  
Cell Types ◽  
S Phase ◽  
Partial Replacement ◽  
Root Tips ◽  
Cell Cycles ◽  
Daughter Cells

ABSTRACTPlant cells undergo two types of cell cycles – the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2’-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Here, we compare sequence-based RT profiles and found that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small, with a median size of 135 kb, and shift to a later RT in the endocycle. However, we found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere in each case, which are ∼1–2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells, but primarily in late S phase of the endocycle. Strikingly, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in nuclei of different ploidies suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and reduced CENH3 enrichment after endocycle replication is consistent with the hypothesis that centromeres are being inactivated as their function is no longer needed.AUTHOR SUMMARYIn traditional cell division, or mitosis, a cell’s genetic material is duplicated and then split between two daughter cells. In contrast, in some specialized cell types, the DNA is duplicated a second time without an intervening division step, resulting in cells that carry twice as much DNA – a phenomenon called an endocycle, which is common during plant development. At each step, DNA replication follows an ordered program, in which highly compacted DNA is unraveled and replicated in sections at different times during the synthesis (S) phase. In plants, it is unclear whether traditional and endocycle programs are the same. Using root tips of maize, we found a small portion of the genome whose replication in the endocycle is shifted in time, usually to later in S phase. Some of these regions are scattered around the genome, and mostly coincide with active genes. However, the most prominent shifts occur in centromeres. This location is noteworthy because centromeres orchestrate the process of separating duplicated chromosomes into daughter cells, a function that is not needed in the endocycle. Our observation that centromeres replicate later in the endocycle suggests there is an important link between the time of replication and the function of centromeres.

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 How MCM loading and spreading specify eukaryotic DNA replication initiation sites

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 2063 ◽  
Author(s):  
Olivier Hyrien
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Origin Recognition Complex ◽  
Cell Types ◽  
S Phase ◽  
Replication Initiation ◽  
Replication Process ◽  
Dna Replication Initiation ◽  
Eukaryotic Dna ◽  
Transcription Complexes

DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

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