scholarly journals Direct binding of Cdt2 to PCNA is important for targeting the CRL4Cdt2 E3 ligase activity to Cdt1

2018 ◽  
Author(s):  
Akiyo Hayashi ◽  
Nickolaos Nikiforos Giakoumakis ◽  
Tatjana Heidebrecht ◽  
Takashi Ishii ◽  
Andreas Panagopoulos ◽  
...  
Keyword(s):  
Genome Stability ◽  
Cell Cycle Progression ◽  
New Paradigm ◽  
Protein Motif ◽  
Cycle Progression ◽  
X Ray ◽  
Ubiquitin Ligase Complex ◽  
Direct Binding ◽  
Crystallographic Structures ◽  
Necessary And Sufficient

AbstractThe CRL4Cdt2 ubiquitin ligase complex is an essential regulator of cell-cycle progression and genome stability, ubiquitinating substrates such as p21, Set8 and Cdt1, via a display of substrate degrons on PCNA. Here, we examine the hierarchy of the ligase and substrate recruitment kinetics onto PCNA at sites of DNA replication. We demonstrate that the C-terminal end of Cdt2 bears a PCNA interaction protein motif (PIP box, Cdt2PIP), which is necessary and sufficient for binding of Cdt2 to PCNA. Cdt2PIP binds PCNA directly with high affinity, two orders of magnitude tighter than the PIP box of Cdt1. X-ray crystallographic structures of PCNA bound to Cdt2PIP and Cdt1PIP show that the peptides occupy all three binding sites of the trimeric PCNA ring. Mutating Cdt2PIP weakens the interaction with PCNA, rendering CRL4Cdt2 less effective in Cdt1 ubiquitination and leading to defects in Cdt1 degradation. The molecular mechanism we present suggests a new paradigm for bringing substrates to the CRL4-type ligase, where the substrate receptor and substrates bind to a common multivalent docking platform to enable subsequent ubiquitination.Summary blurbThe C-terminal end of Cdt2 contains a PIP-box for binding to PCNA to promote CRL4Cdt2 function, creating a new paradigm, where the substrate receptor and substrates bind to a common multivalent docking platform for ubiquitination.

scholarly journals Direct binding of Cdt2 to PCNA is important for targeting the CRL4Cdt2 E3 ligase activity to Cdt1

2018 ◽  
Vol 1 (6) ◽  
pp. e201800238 ◽  
Author(s):  
Akiyo Hayashi ◽  
Nickolaos Nikiforos Giakoumakis ◽  
Tatjana Heidebrecht ◽  
Takashi Ishii ◽  
Andreas Panagopoulos ◽  
...  
Keyword(s):  
Genome Stability ◽  
Cell Cycle Progression ◽  
New Paradigm ◽  
Protein Motif ◽  
Cycle Progression ◽  
Nuclear Antigens ◽  
Ubiquitin Ligase Complex ◽  
Direct Binding ◽  
Necessary And Sufficient ◽  
Proliferating Cell

The CRL4Cdt2 ubiquitin ligase complex is an essential regulator of cell-cycle progression and genome stability, ubiquitinating substrates such as p21, Set8, and Cdt1, via a display of substrate degrons on proliferating cell nuclear antigens (PCNAs). Here, we examine the hierarchy of the ligase and substrate recruitment kinetics onto PCNA at sites of DNA replication. We demonstrate that the C-terminal end of Cdt2 bears a PCNA interaction protein motif (PIP box, Cdt2PIP), which is necessary and sufficient for the binding of Cdt2 to PCNA. Cdt2PIP binds PCNA directly with high affinity, two orders of magnitude tighter than the PIP box of Cdt1. X-ray crystallographic structures of PCNA bound to Cdt2PIP and Cdt1PIP show that the peptides occupy all three binding sites of the trimeric PCNA ring. Mutating Cdt2PIP weakens the interaction with PCNA, rendering CRL4Cdt2 less effective in Cdt1 ubiquitination and leading to defects in Cdt1 degradation. The molecular mechanism we present suggests a new paradigm for bringing substrates to the CRL4-type ligase, where the substrate receptor and substrates bind to a common multivalent docking platform to enable subsequent ubiquitination.

scholarly journals Cell cycle-dependent and independent mating blocks ensure fungal zygote survival and ploidy maintenance

PLoS Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
pp. e3001067
Author(s):  
Aleksandar Vještica ◽  
Melvin Bérard ◽  
Gaowen Liu ◽  
Laura Merlini ◽  
Pedro Junior Nkosi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Genome Stability ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Cell Cycle Reentry ◽  
Mating Behaviors ◽  
Cycle Dependent ◽  
Necessary And Sufficient ◽  
Meiotic Cycle

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins postfusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 not only imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least 2 mechanisms where the zygotic fate imposed by Mei2 and the cell cycle reentry triggered by Mei3 synergize to prevent zygotic mating.

scholarly journals Fungal zygote survival and ploidy maintenance rely on cell cycle-dependent and independent mating blocks

2020 ◽  
Author(s):  
Aleksandar Vještica ◽  
Melvin Bérard ◽  
Gaowen Liu ◽  
Laura Merlini ◽  
Pedro Junior Nkosi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Genome Stability ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Cycle Progression ◽  
Mating Behaviors ◽  
Cycle Dependent ◽  
Necessary And Sufficient ◽  
Meiotic Cycle

AbstractTo ensure genome stability, sexually reproducing organisms require that mating brings together exactly two haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins post-fusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least two mechanisms where the zygotic fate imposed by Mei2 and the cell cycle re-entry triggered by Mei3 synergize to prevent zygotic mating.

scholarly journals The Saccharomyces cerevisiae Anaphase-Promoting Complex Interacts with Multiple Histone-Modifying Enzymes To Regulate Cell Cycle Progression

2010 ◽  
Vol 9 (10) ◽  
pp. 1418-1431 ◽  
Author(s):  
Emma L. Turner ◽  
Mackenzie E. Malo ◽  
Marnie G. Pisclevich ◽  
Megan D. Dash ◽  
Gerald F. Davies ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Chromatin Assembly ◽  
Temperature Sensitive ◽  
Anaphase Promoting Complex ◽  
Cycle Progression ◽  
Ubiquitin Ligase Complex ◽  
Genes Encoding ◽  
Dependent Cell ◽  
Histone Modifying Enzymes

ABSTRACT The anaphase-promoting complex (APC), a large evolutionarily conserved ubiquitin ligase complex, regulates cell cycle progression through mitosis and G1. Here, we present data suggesting that APC-dependent cell cycle progression relies on a specific set of posttranslational histone-modifying enzymes. Multiple APC subunit mutants were impaired in total and modified histone H3 protein content. Acetylated H3K56 (H3K56Ac) levels were as reduced as those of total H3, indicating that loading histones with H3K56Ac is unaffected in APC mutants. However, under restrictive conditions, H3K9Ac and dimethylated H3K79 (H3K79me2) levels were more greatly reduced than those of total H3. In a screen for histone acetyltransferase (HAT) and histone deacetylase (HDAC) mutants that genetically interact with the apc5 CA (chromatin assembly) mutant, we found that deletion of GCN5 or ELP3 severely hampered apc5 CA temperature-sensitive (ts) growth. Further analyses showed that (i) the elp3Δ gcn5Δ double mutant ts defect was epistatic to that observed in apc5 CA cells; (ii) gcn5Δ and elp3Δ mutants accumulate in mitosis; and (iii) turnover of the APC substrate Clb2 is not impaired in elp3Δ gcn5Δ cells. Increased expression of ELP3 and GCN5, as well as genes encoding the HAT Rtt109 and the chromatin assembly factors Msi1 and Asf1, suppressed apc5 CA defects, while increased APC5 expression partially suppressed elp3Δ gcn5Δ growth defects. Finally, we demonstrate that Gcn5 is unstable during G1 and following G1 arrest and is stabilized in APC mutants. We present our working model in which Elp3/Gcn5 and the APC work together to facilitate passage through mitosis and G1. To progress into S, we propose that at least Gcn5 must then be targeted for degradation in an APC-dependent fashion.

scholarly journals PHF2 histone demethylase prevents DNA damage and genome instability by controlling cell cycle progression of neural progenitors

2019 ◽  
Vol 116 (39) ◽  
pp. 19464-19473 ◽  
Author(s):  
Stella Pappa ◽  
Natalia Padilla ◽  
Simona Iacobucci ◽  
Marta Vicioso ◽  
Elena Álvarez de la Campa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Genome Stability ◽  
Cell Cycle Progression ◽  
Genome Instability ◽  
Neural Progenitors ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Biochemical Assays

Histone H3 lysine 9 methylation (H3K9me) is essential for cellular homeostasis; however, its contribution to development is not well established. Here, we demonstrate that the H3K9me2 demethylase PHF2 is essential for neural progenitor proliferation in vitro and for early neurogenesis in the chicken spinal cord. Using genome-wide analyses and biochemical assays we show that PHF2 controls the expression of critical cell cycle progression genes, particularly those related to DNA replication, by keeping low levels of H3K9me3 at promoters. Accordingly, PHF2 depletion induces R-loop accumulation that leads to extensive DNA damage and cell cycle arrest. These data reveal a role of PHF2 as a guarantor of genome stability that allows proper expansion of neural progenitors during development.

Comparative genomics identifies genes mediating cardiotoxicity in the embryonic zebrafish heart

2008 ◽  
Vol 33 (2) ◽  
pp. 148-158 ◽  
Author(s):  
Jing Chen ◽  
Sara A. Carney ◽  
Richard E. Peterson ◽  
Warren Heideman
Keyword(s):  
Heart Failure ◽  
Cell Cycle ◽  
Heart Development ◽  
Cell Cycle Progression ◽  
Transcriptional Response ◽  
Cell Cycle Gene ◽  
Zebrafish Embryos ◽  
Transcriptional Responses ◽  
Cycle Progression ◽  
Necessary And Sufficient

Retinoic acid (RA) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) activate distinct ligand-dependent transcription factors, and both cause cardiac malformation and heart failure in zebrafish embryos. We hypothesized that they cause this response by hyperactivating a common set of genes critical for heart development. To test this, we used microarrays to measure transcript changes in hearts isolated from zebrafish embryos 1, 2, 4, and 12 h after exposure to 1 μM RA. We used hierarchical clustering to compare the transcriptional responses produced in the embryonic heart by RA and TCDD. We could identify no early responses in common between the two agents. However, at 12 h both treatments produced a dramatic downregulation of a common cluster of cell cycle progression genes, which we term the cell cycle gene cluster. This was associated with a halt in heart growth. These results suggest that RA and TCDD ultimately trigger a common transcriptional response associated with heart failure, but not through the direct activation of a common set of genes. Among the genes rapidly induced by RA was Nr2F5, a member of the COUP-TF family of transcriptional repressors. We found that induction of Nr2F5 was both necessary and sufficient for the cardiotoxic response to RA.

Cellular functions of the BRCA tumour-suppressor proteins

2006 ◽  
Vol 34 (5) ◽  
pp. 633-645 ◽  
Author(s):  
S.J. Boulton
Keyword(s):  
Ubiquitin Ligase ◽  
Genome Stability ◽  
Cell Cycle Progression ◽  
Sex Chromosome ◽  
Cellular Level ◽  
Cellular Functions ◽  
Brca1 And Brca2 ◽  
Cycle Progression ◽  
Future Directions ◽  
Meiotic Sex Chromosome Inactivation

Inherited germline mutations in either BRCA1 or BRCA2 confer a significant lifetime risk of developing breast or ovarian cancer. Defining how these two genes function at the cellular level is essential for understanding their role in tumour suppression. Although BRCA1 and BRCA2 were independently cloned over 10 years ago, it is only in the last few years that significant progress has been made towards understanding their function in cells. It is now widely accepted that both genes play critical roles in the maintenance of genome stability. Evidence implicates BRCA2 as an integral component of the homologous recombination machinery, whereas BRCA1 is an E3 ubiquitin ligase that has an impact on DNA repair, transcriptional regulation, cell-cycle progression and meiotic sex chromosome inactivation. In this article, I will review the most recent advances and provide a perspective of potential future directions in this field.

scholarly journals Galectin-1 Induces Reversible Phosphatidylserine Exposure at the Plasma Membrane

2009 ◽  
Vol 20 (5) ◽  
pp. 1408-1418 ◽  
Author(s):  
Sean R. Stowell ◽  
Sougata Karmakar ◽  
Connie M. Arthur ◽  
Tongzhong Ju ◽  
Lilian C. Rodrigues ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Caspase Activation ◽  
New Paradigm ◽  
Mitochondrial Potential ◽  
Cycle Progression ◽  
Membrane Morphology ◽  
Bleb Formation ◽  
Induction Of Apoptosis ◽  
Cellular Turnover

Cells normally undergo physiological turnover through the induction of apoptosis and phagocytic removal, partly through exposure of cell surface phosphatidylserine (PS). In contrast, neutrophils appear to possess apoptosis-independent mechanisms of removal. Here we show that Galectin-1 (Gal-1) induces PS exposure independent of alterations in mitochondrial potential, caspase activation, or cell death. Furthermore, Gal-1–induced PS exposure reverts after Gal-1 removal without altering cell viability. Gal-1–induced PS exposure is uniquely microdomain restricted, yet cells exposing PS do not display evident alterations in membrane morphology nor do they exhibit bleb formation, typically seen in apoptotic cells. Long-term exposure to Gal-1 prolongs PS exposure with no alteration in cell cycle progression or cell growth. These results demonstrate that Gal-1–induced PS exposure and subsequent phagocytic removal of living cells represents a new paradigm in cellular turnover.

scholarly journals In vitro Assessment of the DNA Damage Response in Dental Mesenchymal Stromal Cells Following Low Dose X-ray Exposure

2021 ◽  
Vol 9 ◽  
Author(s):  
Niels Belmans ◽  
Liese Gilles ◽  
Jonas Welkenhuysen ◽  
Randy Vermeesen ◽  
Bjorn Baselet ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Cell Cycle Progression ◽  
Deciduous Teeth ◽  
Low Doses ◽  
Cycle Progression ◽  
X Ray

Stem cells contained within the dental mesenchymal stromal cell (MSC) population are crucial for tissue homeostasis. Assuring their genomic stability is therefore essential. Exposure of stem cells to ionizing radiation (IR) is potentially detrimental for normal tissue homeostasis. Although it has been established that exposure to high doses of ionizing radiation (IR) has severe adverse effects on MSCs, knowledge about the impact of low doses of IR is lacking. Here we investigated the effect of low doses of X-irradiation with medical imaging beam settings (<0.1 Gray; 900 mGray per hour), in vitro, on pediatric dental mesenchymal stromal cells containing dental pulp stem cells from deciduous teeth, dental follicle progenitor cells and stem cells from the apical papilla. DNA double strand break (DSB) formation and repair kinetics were monitored by immunocytochemistry of γH2AX and 53BP1 as well as cell cycle progression by flow cytometry and cellular senescence by senescence-associated β-galactosidase assay and ELISA. Increased DNA DSB repair foci, after exposure to low doses of X-rays, were measured as early as 30 min post-irradiation. The number of DSBs returned to baseline levels 24 h after irradiation. Cell cycle analysis revealed marginal effects of IR on cell cycle progression, although a slight G2/M phase arrest was seen in dental pulp stromal cells from deciduous teeth 72 h after irradiation. Despite this cell cycle arrest, no radiation-induced senescence was observed. In conclusion, low X-ray IR doses (< 0.1 Gray; 900 mGray per hour), were able to induce significant increases in the number of DNA DSBs repair foci, but cell cycle progression seems to be minimally affected. This highlights the need for more detailed and extensive studies on the effects of exposure to low IR doses on different mesenchymal stromal cells.

scholarly journals p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Longtao Wu ◽  
Clayton D. Crawley ◽  
Andrea Garofalo ◽  
Jackie W. Nichols ◽  
Paige-Ashley Campbell ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Genome Stability ◽  
Cell Cycle Progression ◽  
Human Cancer ◽  
S Phase ◽  
Post Translational Modification ◽  
Chromosomal Breakage ◽  
Cycle Progression ◽  
Genome Wide ◽  
Phase Progression

Abstract p50, the mature product of NFKB1, is constitutively produced from its precursor, p105. Here, we identify BARD1 as a p50-interacting factor. p50 directly associates with the BARD1 BRCT domains via a C-terminal phospho-serine motif. This interaction is induced by ATR and results in mono-ubiquitination of p50 by the BARD1/BRCA1 complex. During the cell cycle, p50 is mono-ubiquitinated in S phase and loss of this post-translational modification increases S phase progression and chromosomal breakage. Genome-wide studies reveal a substantial decrease in p50 chromatin enrichment in S phase and Cycln E is identified as a factor regulated by p50 during the G1 to S transition. Functionally, interaction with BARD1 promotes p50 protein stability and consistent with this, in human cancer specimens, low nuclear BARD1 protein strongly correlates with low nuclear p50. These data indicate that p50 mono-ubiquitination by BARD1/BRCA1 during the cell cycle regulates S phase progression to maintain genome integrity.

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