Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level

Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.

Download Full-text

Fusion Tyrosine Kinases Induce Drug Resistance by Stimulation of Homology-Dependent Recombination Repair, Prolongation of G2/M Phase, and Protection from Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.22.12.4189-4201.2002 ◽

2002 ◽

Vol 22 (12) ◽

pp. 4189-4201 ◽

Cited By ~ 146

Author(s):

Artur Slupianek ◽

Grazyna Hoser ◽

Ireneusz Majsterek ◽

Agnieszka Bronisz ◽

Maciej Malecki ◽

...

Keyword(s):

Dna Damage ◽

Drug Resistance ◽

Tyrosine Kinases ◽

Transformed Cells ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Recombination Repair ◽

M Phase ◽

Induced Apoptosis ◽

In Cells

ABSTRACT Fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFβR, TEL/TRKC(L), and NPM/ALK arise from reciprocal chromosomal translocations and cause acute and chronic leukemias and non-Hodgkin's lymphoma. FTK-transformed cells displayed drug resistance against the cytostatic drugs cisplatin and mitomycin C. These cells were not protected from drug-mediated DNA damage, implicating activation of the mechanisms preventing DNA damage-induced apoptosis. Various FTKs, except TEL/TRKC(L), can activate STAT5, which may be required to induce drug resistance. We show that STAT5 is essential for FTK-dependent upregulation of RAD51, which plays a central role in homology-dependent recombinational repair (HRR) of DNA double-strand breaks (DSBs). Elevated levels of Rad51 contributed to the induction of drug resistance and facilitation of the HRR in FTK-transformed cells. In addition, expression of antiapoptotic protein Bcl-xL was enhanced in cells transformed by the FTKs able to activate STAT5. Moreover, cells transformed by all examined FTKs displayed G2/M delay upon drug treatment. Individually, elevated levels of Rad51, Bcl-xL, or G2/M delay were responsible for induction of a modest drug resistance. Interestingly, combination of these three factors in nontransformed cells induced drug resistance of a magnitude similar to that observed in cells expressing FTKs activating STAT5. Thus, we postulate that RAD51-dependent facilitation of DSB repair, antiapoptotic activity of Bcl-xL, and delay in progression through the G2/M phase work in concert to induce drug resistance in FTK-positive leukemias and lymphomas.

Download Full-text

Multiple autophosphorylation sites are dispensable for murine ATM activation in vivo

The Journal of Cell Biology ◽

10.1083/jcb.200805154 ◽

2008 ◽

Vol 183 (5) ◽

pp. 777-783 ◽

Cited By ~ 59

Author(s):

Jeremy A. Daniel ◽

Manuela Pellegrini ◽

Ji-Hoon Lee ◽

Tanya T. Paull ◽

Lionel Feigenbaum ◽

...

Keyword(s):

Dna Damage ◽

Cell Cycle Checkpoints ◽

Activation Mechanism ◽

Mutant Form ◽

Dna Double Strand Breaks ◽

Atm Kinase ◽

Strand Breaks ◽

Physiological Importance ◽

Evolutionarily Conserved

Cellular responses to both physiological and pathological DNA double-strand breaks are initiated through activation of the evolutionarily conserved ataxia telangiectasia mutated (ATM) kinase. Upon DNA damage, an activation mechanism involving autophosphorylation has been reported to allow ATM to phosphorylate downstream targets important for cell cycle checkpoints and DNA repair. In humans, serine residues 367, 1893, and 1981 have been shown to be autophosphorylation sites that are individually required for ATM activation. To test the physiological importance of these sites, we generated a transgenic mouse model in which all three conserved ATM serine autophosphorylation sites (S367/1899/1987) have been replaced with alanine. In this study, we show that ATM-dependent responses at both cellular and organismal levels are functional in mice that express a triple serine mutant form of ATM as their sole ATM species. These results lend further support to the notion that ATM autophosphorylation correlates with the DNA damage–induced activation of the kinase but is not required for ATM function in vivo.

Download Full-text

Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis

BioMed Research International ◽

10.1155/2016/8279560 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 1

Author(s):

Teresa Anglada ◽

Mariona Terradas ◽

Laia Hernández ◽

Anna Genescà ◽

Marta Martín

Keyword(s):

Dna Damage ◽

Ataxia Telangiectasia ◽

Dna Double Strand Breaks ◽

Predictive Tool ◽

Normal Cells ◽

Strand Breaks ◽

Radiation Induced ◽

Late Apoptosis ◽

Induced Apoptosis ◽

The Relationship

In order to examine the relationship between accumulation of residual DNA double-strand breaks (DSBs) and cell death, we have used a control and an ATM (Ataxia-Telangiectasia Mutated) defective cell line, as Ataxia-Telangiectasia (AT) cells tend to accumulate residual DSBs at long times after damage infliction. After irradiation, AT cells showed checkpoint impairment and a fraction of cells displayed an abnormal centrosome number and tetraploid DNA content, and this fraction increased along with apoptosis rates. At all times analyzed, AT cells displayed a significantly higher rate of radiation-induced apoptosis than normal cells. Besides apoptosis, 70–85% of the AT viable cells (TUNEL-negative) carried ≥10γH2AX foci/cell, while only 12–27% of normal cells did. The fraction of AT and normal cells undergoing early and late apoptosis were isolated by flow cytometry and residual DSBs were concretely scored in these populations. Half of theγH2AX-positive AT cells undergoing early apoptosis carried ≥10γH2AX foci/cell and this fraction increased to 75% in late apoptosis. The results suggest that retention of DNA damage-inducedγH2AX foci is an indicative of lethal DNA damage, as cells undergoing apoptosis are those accumulating more DSBs. Scoring of residualγH2AX foci might function as a predictive tool to assess radiation-induced apoptosis.

Download Full-text

Impeding DNA Break Repair Enables Oocyte Quality Control

10.1101/277913 ◽

2018 ◽

Cited By ~ 1

Author(s):

Huanyu Qiao ◽

H.B.D. Prasada Rao ◽

Yan Yun ◽

Sumit Sandhu ◽

Jared H. Fong ◽

...

Keyword(s):

Quality Control ◽

Oocyte Quality ◽

Double Strand Breaks ◽

Negative Regulator ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Chromosome Synapsis ◽

Sumo Ligase ◽

Induced Apoptosis

SUMMARYOocyte quality control culls eggs with defects in meiosis. In mouse, oocyte death is triggered by defects in chromosome synapsis and recombination, which involve repair of programmed DNA double-strand breaks (DSBs) between homologous chromosomes. We show that RNF212, a SUMO ligase required for crossing over, also mediates oocyte quality control. Both physiological apoptosis and wholesale oocytes elimination in meiotic mutants require RNF212. RNF212 sensitizes cells to DSB-induced apoptosis within a narrow window when chromosomes desynapse during the transition into quiescence. Analysis of DNA damage during this transition implies that RNF212 impedes DSB repair. Consistently, RNF212 is required for HORMAD1, a negative regulator of inter-sister recombination, to associate with desynapsing chromosomes. We infer that oocytes impede repair of residual DSBs to retain a “memory” of meiotic defects that enables quality control processes. These results define the logic of oocyte quality control and suggest RNF212 variants may influence transmission of defective genomes.

Download Full-text

DNA damage interactions on both nanometer and micrometer scale determine overall cellular damage

Scientific Reports ◽

10.1038/s41598-018-34323-9 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 16

Author(s):

Thomas Friedrich ◽

Katarina Ilicic ◽

Christoph Greubel ◽

Stefanie Girst ◽

Judith Reindl ◽

...

Keyword(s):

Dna Damage ◽

Cellular Level ◽

Dna Lesions ◽

Double Strand Breaks ◽

Cellular Damage ◽

Biophysical Model ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Dna Lesion ◽

Micrometer Scale

Abstract DNA double strand breaks (DSB) play a pivotal role for cellular damage, which is a hazard encountered in toxicology and radiation protection, but also exploited e.g. in eradicating tumors in radiation therapy. It is still debated whether and in how far clustering of such DNA lesions leads to an enhanced severity of induced damage. Here we investigate - using focused spots of ionizing radiation as damaging agent - the spatial extension of DNA lesion patterns causing cell inactivation. We find that clustering of DNA damage on both the nm and µm scale leads to enhanced inactivation compared to more homogeneous lesion distributions. A biophysical model interprets these observations in terms of enhanced DSB production and DSB interaction, respectively. We decompose the overall effects quantitatively into contributions from these lesion formation processes, concluding that both processes coexist and need to be considered for determining the resulting damage on the cellular level.

Download Full-text

PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome

Nature Communications ◽

10.1038/s41467-019-13641-0 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 2

Author(s):

Deepti Sharma ◽

Louis De Falco ◽

Sivaraman Padavattan ◽

Chang Rao ◽

Susana Geifman-Shochat ◽

...

Keyword(s):

Dna Damage ◽

Mutational Analysis ◽

Catalytic Efficiency ◽

Double Strand Breaks ◽

Genomic Integrity ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Nucleosome Core ◽

Epigenetic Marker ◽

Kinetics Of

AbstractThe poly(ADP-ribose) polymerase, PARP1, plays a key role in maintaining genomic integrity by detecting DNA damage and mediating repair. γH2A.X is the primary histone marker for DNA double-strand breaks and PARP1 localizes to H2A.X-enriched chromatin damage sites, but the basis for this association is not clear. We characterize the kinetics of PARP1 binding to a variety of nucleosomes harbouring DNA double-strand breaks, which reveal that PARP1 associates faster with (γ)H2A.X- versus H2A-nucleosomes, resulting in a higher affinity for the former, which is maximal for γH2A.X-nucleosome that is also the activator eliciting the greatest poly-ADP-ribosylation catalytic efficiency. The enhanced activities with γH2A.X-nucleosome coincide with increased accessibility of the DNA termini resulting from the H2A.X-Ser139 phosphorylation. Indeed, H2A- and (γ)H2A.X-nucleosomes have distinct stability characteristics, which are rationalized by mutational analysis and (γ)H2A.X-nucleosome core crystal structures. This suggests that the γH2A.X epigenetic marker directly facilitates DNA repair by stabilizing PARP1 association and promoting catalysis.

Download Full-text

RepID-deficient cancer cells are sensitized to a drug targeting p97/VCP segregase

Molecular & Cellular Toxicology ◽

10.1007/s13273-021-00121-0 ◽

2021 ◽

Author(s):

Sang-Min Jang ◽

Christophe E. Redon ◽

Haiqing Fu ◽

Fred E. Indig ◽

Mirit I. Aladjem

Keyword(s):

Dna Damage ◽

Cancer Cells ◽

Cell Sorting ◽

Clonogenic Survival ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Ubiquitinated Proteins ◽

Damage Response ◽

Survival Assay

Abstract Background The p97/valosin-containing protein (VCP) complex is a crucial factor for the segregation of ubiquitinated proteins in the DNA damage response and repair pathway. Objective We investigated whether blocking the p97/VCP function can inhibit the proliferation of RepID-deficient cancer cells using immunofluorescence, clonogenic survival assay, fluorescence-activated cell sorting, and immunoblotting. Result p97/VCP was recruited to chromatin and colocalized with DNA double-strand breaks in RepID-deficient cancer cells that undergo spontaneous DNA damage. Inhibition of p97/VCP induced death of RepID-depleted cancer cells. This study highlights the potential of targeting p97/VCP complex as an anticancer therapeutic approach. Conclusion Our results show that RepID is required to prevent excessive DNA damage at the endogenous levels. Localization of p97/VCP to DSB sites was induced based on spontaneous DNA damage in RepID-depleted cancer cells. Anticancer drugs targeting p97/VCP may be highly potent in RepID-deficient cells. Therefore, we suggest that p97/VCP inhibitors synergize with RepID depletion to kill cancer cells.

Download Full-text

Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy

International Journal of Molecular Sciences ◽

10.3390/ijms22147638 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7638

Author(s):

Yvonne Lorat ◽

Judith Reindl ◽

Anna Isermann ◽

Christian Rübe ◽

Anna A. Friedl ◽

...

Keyword(s):

Electron Microscopy ◽

Dna Damage ◽

Spatial Clustering ◽

Dna Lesions ◽

Double Strand Breaks ◽

Stimulated Emission ◽

Nuclear Chromatin ◽

Carbon Ions ◽

Dna Double Strand Breaks ◽

Strand Breaks

Background: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. Material and Methods: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). Results: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. Conclusion: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

Download Full-text

Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906102116 ◽

2019 ◽

Vol 116 (39) ◽

pp. 19552-19562 ◽

Cited By ~ 7

Author(s):

Justine Sitz ◽

Sophie Anne Blanchet ◽

Steven F. Gameiro ◽

Elise Biquand ◽

Tia M. Morgan ◽

...

Keyword(s):

Cervical Cancer ◽

Dna Damage ◽

Cancer Cells ◽

Genomic Instability ◽

E3 Ligase ◽

Human Papillomaviruses ◽

Double Strand Breaks ◽

Nuclear Bodies ◽

Dna Double Strand Breaks ◽

Strand Breaks

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell’s DNA replication and repair machineries to replicate their own genomes. How this host–pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

Download Full-text

Focused ultrasound radiosensitizes human cancer cells by enhancement of DNA damage

Strahlentherapie und Onkologie ◽

10.1007/s00066-021-01774-5 ◽

2021 ◽

Author(s):

Xinrui Zhang ◽

Mariana Bobeica ◽

Michael Unger ◽

Anastasia Bednarz ◽

Bjoern Gerold ◽

...

Keyword(s):

Prostate Cancer ◽

Dna Damage ◽

Cancer Cells ◽

Metabolic Activity ◽

Focused Ultrasound ◽

Double Strand Breaks ◽

High Intensity Focused Ultrasound ◽

Dna Double Strand Breaks ◽

Strand Breaks

Abstract Purpose High-intensity focused ultrasound (HIFU/FUS) has expanded as a noninvasive quantifiable option for hyperthermia (HT). HT in a temperature range of 40–47 °C (thermal dose CEM43 ≥ 25) could work as a sensitizer to radiation therapy (RT). Here, we attempted to understand the tumor radiosensitization effect at the cellular level after a combination treatment of FUS+RT. Methods An in vitro FUS system was developed to induce HT at frequencies of 1.147 and 1.467 MHz. Human head and neck cancer (FaDU), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS in ultrasound-penetrable 96-well plates followed by single-dose X‑ray irradiation (10 Gy). Radiosensitizing effects of FUS were investigated by cell metabolic activity (WST‑1 assay), apoptosis (annexin V assay, sub-G1 assay), cell cycle phases (propidium iodide staining), and DNA double-strand breaks (γH2A.X assay). Results The FUS intensities of 213 (1.147 MHz) and 225 W/cm2 (1.467 MHz) induced HT for 30 min at mean temperatures of 45.20 ± 2.29 °C (CEM43 = 436 ± 88) and 45.59 ± 1.65 °C (CEM43 = 447 ± 79), respectively. FUS improves the effect of RT significantly by reducing metabolic activity in T98G cells 48 h (RT: 96.47 ± 8.29%; FUS+RT: 79.38 ± 14.93%; p = 0.012) and in PC-3 cells 72 h (54.20 ± 10.85%; 41.01 ± 11.17%; p = 0.016) after therapy, but not in FaDu cells. Mechanistically, FUS+RT leads to increased apoptosis and enhancement of DNA double-strand breaks compared to RT alone in T98G and PC-3 cells. Conclusion Our in vitro findings demonstrate that FUS has good potential to sensitize glioblastoma and prostate cancer cells to RT by mainly enhancing DNA damage.

Download Full-text