Oocyte Elimination Through DNA Damage Signaling from CHK1/CHK2 to p53 and p63

Eukaryotic organisms have evolved mechanisms to prevent the accumulation of cells bearing genetic aberrations. This is especially crucial for the germline, because fecundity and fitness of progeny would be adversely affected by an excessively high mutational incidence. The process of meiosis poses unique problems for mutation avoidance because of the requirement for SPO11-induced programmed double-strand breaks (DSBs) in recombination-driven pairing and segregation of homologous chromosomes. Mouse meiocytes bearing unrepaired meiotic DSBs or unsynapsed chromosomes are eliminated before completing meiotic prophase I. In previous work, we showed that checkpoint kinase 2 (CHK2; CHEK2), a canonical DNA damage response protein, is crucial for eliminating not only oocytes defective in meiotic DSB repair (e.g., Trip13Gt mutants), but also Spo11−/− oocytes that are defective in homologous chromosome synapsis and accumulate a threshold level of spontaneous DSBs. However, rescue of such oocytes by Chk2 deficiency was incomplete, raising the possibility that a parallel checkpoint pathway(s) exists. Here, we show that mouse oocytes lacking both p53 (TRP53) and the oocyte-exclusive isoform of p63, TAp63, protects nearly all Spo11−/− and Trip13Gt/Gt oocytes from elimination. We present evidence that checkpoint kinase I (CHK1; CHEK1), which is known to signal to TRP53, also becomes activated by persistent DSBs in oocytes, and to an increased degree when CHK2 is absent. The combined data indicate that nearly all oocytes reaching a threshold level of unrepaired DSBs are eliminated by a semiredundant pathway of CHK1/CHK2 signaling to TRP53/TAp63.

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Signaling to TRP53 and TAp63 from CHK1/CHK2 is responsible for elimination of most oocytes defective for either chromosome synapsis or recombination

10.1101/768150 ◽

2019 ◽

Author(s):

Vera D. Rinaldi ◽

Jordana C. Bloom ◽

John C. Schimenti

Keyword(s):

Threshold Level ◽

Double Strand Breaks ◽

Checkpoint Kinase ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Chromosome Synapsis ◽

Checkpoint Pathway ◽

Mutation Avoidance ◽

Meiotic Dsbs ◽

Eukaryotic Organisms

ABSTRACTEukaryotic organisms have evolved mechanisms to prevent the accumulation of cells bearing genetic aberrations. This is especially crucial for the germline, because fecundity, and fitness of progeny would be adversely affected by an excessively high mutational incidence. The process of meiosis poses unique problems for mutation avoidance, due to the requirement for SPO11-induced programmed double strand breaks (DSBs) in recombination-driven pairing and segregation of homologous chromosomes. Mouse meiocytes bearing unrepaired meiotic DSBs or unsynapsed chromosomes are eliminated before completing meiotic prophase I. In previous work, we showed that checkpoint kinase 2 (CHK2; CHEK2), a canonical DNA damage response protein, is crucial for eliminating not only oocytes defective in meiotic DSB repair (e.g. Trip13Gt mutants), but also asynaptic Spo11−/− oocytes that accumulate a threshold level of spontaneous DSBs. However, rescue of such oocytes by Chk2 deficiency was incomplete, raising the possibility that a parallel checkpoint pathway(s) exists. Here, we show that mouse oocytes lacking both TAp63 and TRP53 protects nearly all Spo11−/− and Trip13Gt/Gt oocytes from elimination. We present evidence that checkpoint kinase I (CHK1; CHEK1), which is known to signal to TRP53, also becomes activated by persistent DSBs in oocytes, and to an increased degree when CHK2 is absent. The combined data indicate that nearly all oocytes reaching a threshold level of unrepaired DSBs are eliminated by a semi-redundant pathway of CHK1/CHK2 signaling to TRP53/TAp63.

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A single checkpoint pathway eliminates mouse oocytes with dna damage or chromosome synapsis failure

10.1101/137075 ◽

2017 ◽

Author(s):

Vera D. Rinaldi ◽

Ewelina Bolcun-Filas ◽

Hiroshi Kogo ◽

Hiroki Kurahashi ◽

John C. Schimenti

Keyword(s):

Dna Damage ◽

Substantial Fraction ◽

Activation Threshold ◽

Late Prophase ◽

Checkpoint Activation ◽

Homologous Chromosomes ◽

Mouse Oocytes ◽

Chromosome Synapsis ◽

Checkpoint Pathway ◽

Meiotic Dsbs

Pairing and synapsis of homologous chromosomes during meiosis is crucial for producing genetically normal gametes, and is dependent upon repair of SPO11-induced double stranded breaks (DSBs) by homologous recombination. To prevent transmission of genetic defects, diverse organisms have evolved mechanisms to eliminate meiocytes containing unrepaired DSBs or unsynapsed chromosomes. Here, we show that the CHK2 (CHEK2)-dependent DNA damage checkpoint culls not only recombination-defective mouse oocytes, but also SPO11-deficient oocytes that are severely defective in homolog synapsis. The checkpoint is triggered by spontaneous DSBs that arise in late prophase I, accumulating above the checkpoint activation threshold (∼10 DSBs) because presence of HORMAD1/2 on unsynapsed chromosome axes prevents their repair. Furthermore, Hormad2 deletion rescued fertility and meiotic DSB repair of oocytes containing a synapsis-proficient, non-crossover recombination defective mutation in a gene (Trip13) required for removal of HORMADs from synapsed chromosomes, indicating that a substantial fraction of meiotic DSBs are normally repaired by intersister recombination in mice.

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STK38 promotes ATM activation by acting as a reader of histone H4 ufmylation

Science Advances ◽

10.1126/sciadv.aax8214 ◽

2020 ◽

Vol 6 (23) ◽

pp. eaax8214 ◽

Cited By ~ 1

Author(s):

Bo Qin ◽

Jia Yu ◽

Somaira Nowsheen ◽

Fei Zhao ◽

Liewei Wang ◽

...

Keyword(s):

Dna Damage ◽

Hippo Pathway ◽

Double Strand Breaks ◽

Binding Motif ◽

Histone H4 ◽

Ataxia Telangiectasia Mutated ◽

Independent Manner ◽

Strand Breaks ◽

Dna Damage Signaling ◽

The Hippo Pathway

The ATM (ataxia-telangiectasia mutated) kinase is rapidly activated following DNA damage and phosphorylates its downstream targets to launch DDR signaling. Recently, we and others showed that UFM1 signaling promotes ATM activation. We further discovered that monoufmylation of histone H4 at Lys31 by UFM1-specific ligase 1 (UFL1) is an important step in the amplification of ATM activation. However, how monoufmylated H4 enhances ATM activation is still unknown. Here, we report STK38, a kinase in the Hippo pathway, serves as a reader for histone H4 ufmylation to promote ATM activation in a kinase-independent manner. STK38 contains a potential UFM1 binding motif which recognizes ufmylated H4 and recruits the SUV39H1 to the double-strand breaks, resulting in H3K9 trimethylation and Tip60 activation to promote ATM activation. Together, STK38 is a previously unknown player in DNA damage signaling and functions as a reader of monoufmylated H4 at Lys31 to promote ATM activation.

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Tex19.1 Regulates Meiotic DNA Double Strand Break Frequency in Mouse Spermatocytes

10.1101/099119 ◽

2017 ◽

Cited By ~ 4

Author(s):

James H. Crichton ◽

Christopher J. Playfoot ◽

Marie MacLennan ◽

David Read ◽

Howard J. Cooke ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Meiotic Recombination ◽

Homologous Chromosome ◽

E3 Ubiquitin Ligase ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Genetic Pathway ◽

Strand Breaks ◽

Chromosome Synapsis ◽

Meiotic Dsbs

AbstractMeiosis relies on the SPO11 endonuclease to generate the recombinogenic DNA double strand breaks (DSBs) required for homologous chromosome synapsis and segregation. The number of meiotic DSBs needs to be sufficient to allow chromosomes to search for and find their homologs, but not excessive to the point of causing genome instability. Here we report that meiotic DSB frequency in mouse spermatocytes is regulated by the mammal-specific gene Tex19.1. We show that the chromosome asynapsis previously reported in Tex19.1-/- spermatocytes is preceded by reduced numbers of recombination foci in leptotene and zygotene. Tex19.1 is required for the generation of normal levels of Spo11-dependent DNA damage during leptotene, but not for upstream events such as MEI4 foci formation or accumulation of H3K4me3 at recombination hotspots. Furthermore, we show that mice carrying mutations in the E3 ubiquitin ligase UBR2, a TEX19.1-interacting partner, phenocopy the Tex19.1-/- recombination defects. These data show that Tex19.1 and Ubr2 are required for mouse spermatocytes to generate sufficient meiotic DSBs to ensure that homology search is consistently successful, and reveal a hitherto unknown genetic pathway regulating meiotic DSB frequency in mammals.Author SummaryMeiosis is a specialised type of cell division that occurs during sperm and egg development to reduce chromosome number prior to fertilisation. Recombination is a key step in meiosis as it facilitates the pairing of homologous chromosomes prior to their reductional division, and generates new combinations of genetic alleles for transmission in the next generation. Regulating the amount of recombination is key for successful meiosis: too much will likely cause mutations, chromosomal re-arrangements and genetic instability, whereas too little causes defects in homologous chromosome pairing prior to the meiotic divisions. This study identifies a genetic pathway requiredto generate robust meiotic recombination in mouse spermatocytes. We show that male mice with mutations in Tex19.1 or Ubr2, which encodes an E3 ubiquitin ligase that interacts with TEX19.1, have defects in generating normal levels of meiotic recombination. We show that the defects in these mutants impact on the recombination process at the stage when programmed DNA double strand breaks are being made. This defect likely contributes to the chromosome synapsis and meiotic progression phenotypes previously described in these mutant mice. This study has implications for our understanding of how this fundamental aspect of genetics and inheritance is controlled.

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ATR is required to complete meiotic recombination in mice

10.1101/133744 ◽

2017 ◽

Cited By ~ 3

Author(s):

Sarai Pacheco ◽

Andros Maldonado-Linares ◽

Marina Marcet-Ortega ◽

Cristina Rojas ◽

Ana Martínez-Marchal ◽

...

Keyword(s):

Meiotic Recombination ◽

Meiotic Prophase ◽

Protein A ◽

Homology Search ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Molecular Features ◽

Chromosome Synapsis ◽

Meiotic Dsbs

ABSTRACTPrecise execution of recombination during meiosis is essential for forming chromosomally balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Binding of replication protein A (RPA) at resected DSBs fosters association of RAD51 and DMC1, the primary effectors of homology search. It is well appreciated that cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these responses remain poorly understood, particularly in mammals. Here we provide evidence that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.

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SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint

eLife ◽

10.7554/elife.02482 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 121

Author(s):

Sílvia Carvalho ◽

Alexandra C Vítor ◽

Sreerama C Sridhara ◽

Filipa B Martins ◽

Ana C Raposo ◽

...

Keyword(s):

Dna Damage ◽

Strand Break ◽

Dna Lesions ◽

Alternative Mechanism ◽

Dna Double Strand Breaks ◽

Cell Renal Cell Carcinoma ◽

Strand Breaks ◽

Dna Damage Signaling ◽

Dna Double Strand Break ◽

Recombination Repair

Histone modifications establish the chromatin states that coordinate the DNA damage response. In this study, we show that SETD2, the enzyme that trimethylates histone H3 lysine 36 (H3K36me3), is required for ATM activation upon DNA double-strand breaks (DSBs). Moreover, we find that SETD2 is necessary for homologous recombination repair of DSBs by promoting the formation of RAD51 presynaptic filaments. In agreement, SETD2-mutant clear cell renal cell carcinoma (ccRCC) cells displayed impaired DNA damage signaling. However, despite the persistence of DNA lesions, SETD2-deficient cells failed to activate p53, a master guardian of the genome rarely mutated in ccRCC and showed decreased cell survival after DNA damage. We propose that this novel SETD2-dependent role provides a chromatin bookmarking instrument that facilitates signaling and repair of DSBs. In ccRCC, loss of SETD2 may afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for additional genetic mutations in TP53.

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ANKHD1 Silencing Delays S Phase Progression in Multiple Myeloma Cells Via Activation of ATM/ATR -CDC25a Pathway

Blood ◽

10.1182/blood.v128.22.5624.5624 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5624-5624

Author(s):

Dhyani Anamika ◽

Patricia Favaro ◽

Sara Teresinha Olalla Saad

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Dna Damage ◽

S Phase ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Myeloma Cells ◽

Checkpoint Kinase ◽

Strand Breaks ◽

Phase Progression

Abstract Ankyrin repeat and KH domain-containing protein 1, ANKHD1, is highly expressed in myeloma cells and plays an important role in multiple myeloma (MM) progression and growth. ANKHD1 is found to be overexpressed in S phase of cell cycle in MM cells and silencing of ANKHD1 expression leads to accumulation of cells in S phase, suggesting a role in S phase progression (1). Earlier studies by our group reported that ANKHD1 silencing downregulates all replication dependent histones and that this downregulation may be associated with replication stress and DNA damage (2). We observed increased expression of γH2AX protein (phosphorylated histone H2A variant, H2AX, at Serine 139), a marker for DNA double strand breaks (DSBs) and an early sign of DNA damage induced by replication stress, in ANKHD1 silenced MM cells. In the present study we further sought to investigate the mechanisms underlying the induction of DNA damage on ANKHD1 silencing. We first confirmed the increased expression of γH2AX by flow cytometry analysis and observed that both the mean fluorescence intensity as well as percentage of γH2AX positive cells were higher in ANKHD1 silenced MM cells as compared to control cells. Phosphorylation of histone 2AX requires activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated)andATR (ATM-Rad3-related) that serves as central components of the signaling cascade initiated by DSBs. Hence, we checked for the expression of these kinases and observed increased phosphorylation of both ATM and ATR kinases in ANKHD1 silenced MM cells. There was no difference in the expressions of DNA-PKcs in control and ANKHD1 silenced cells by western blot. We next checked for the expression of CHK1 (checkpoint kinase 1) and CHK2 (checkpoint kinase 2), essential serine threonine kinases downstream of ATM and ATR. We observed a decrease in pCHK2 (phosphorylated CHK2 at Thr 68), with no change in expression of pCHK1 (phosphorylated CHK1 at Ser 345) total CHK1 or total CHK2. We also checked for expression of CDC25a (a member of the CDC25 family of dual-specificity phosphatases), that is specifically degraded in response to DNA damage (DSBs) and delays S phase progression via activation of ATM /ATR-CHK2 signaling pathway. Expression of CDC25a was significantly decreased in ANKHD1 silencing cells, confirming the induction of DSBs, and probably accounting for S phase delay on ANKHD1 silencing. Since there was decrease in active CHK2 (pCHK2) and no change in CHK1 required for degradation of CDC25a, we assume that decrease in CDC25a in ANKHD1 silenced MM cells may be via activation of ATM/ ATR pathway independent of CHK2/CHK1. Expression of several other downstream factors of DSBs induced DNA damage response and repair such as BRCA1, PTEN, DNMT1, SP1, HDAC2 were also found to be modulated in ANKHD1 silenced MM cells. In conclusion, ANKHD1 silencing in MM cells leads to DNA damage and modulates expression of several genes implicated in DNA damage and repair. DNA damage induced after ANKHD1 silencing in MM cells activates ATM/ ATR-CDC25a pathway which may lead to the activation of S phase checkpoint in MM cells. Results however are preliminary and further studies are required to understand the role of ANKHD1 in intra S phase check point. References: 1) ANKHD1 regulates cell cycle progression and proliferation in multiple myeloma cells. Dhyani et al. FEBS letters 2012; 586: 4311-18. 2) ANKHD1 is essential for repair of DNA double strand breaks in multiple myeloma. Dhyani et al. ASH Abstract, Blood 2015; 126:1762. Disclosures No relevant conflicts of interest to declare.

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Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication

Journal of Virology ◽

10.1128/jvi.00936-16 ◽

2016 ◽

Vol 90 (20) ◽

pp. 9433-9445 ◽

Cited By ~ 12

Author(s):

Thomas J. Kucharski ◽

Timothy F. Ng ◽

David M. Sharon ◽

Pedram Navid-Azarbaijani ◽

Mahvash Tavassoli ◽

...

Keyword(s):

Dna Damage ◽

Tumor Cell ◽

Tumor Cells ◽

Chicken Anemia Virus ◽

Transformed Cells ◽

Normal Cells ◽

Checkpoint Kinase ◽

Checkpoint Kinase 1 ◽

Dna Damage Signaling ◽

Anemia Virus

ABSTRACTChicken anemia virus (CAV) is a single-stranded circular DNA virus that carries 3 genes, the most studied of which is the gene encoding VP3, also known as apoptin. This protein has been demonstrated to specifically kill transformed cells while leaving normal cells unharmed in a manner that is independent of p53 status. Although the mechanistic basis for this differential activity is unclear, it is evident that the subcellular localization of the protein is important for the difference. In normal cells, apoptin exists in filamentous networks in the cytoplasm, whereas in transformed cells, apoptin is present in the nucleus and appears as distinct foci. We have previously demonstrated that DNA damage signaling through the ataxia telangiectasia mutated (ATM) pathway induces the translocation of apoptin from the cytoplasm to the nucleus, where it induces apoptosis. We found that apoptin contains four checkpoint kinase consensus sites and that mutation of either threonine 56 or 61 to alanine restricts apoptin to the cytoplasm. Furthermore, treatment of tumor cells expressing apoptin with inhibitors of checkpoint kinase 1 (Chk1) and Chk2 causes apoptin to localize to the cytoplasm. Importantly, silencing of Chk2 rescues cancer cells from the cytotoxic effects of apoptin. Finally, treatment of virus-producing cells with Chk inhibitor protects them from virus-mediated toxicity and reduces the titer of progeny virus. Taken together, our results indicate that apoptin is a sensor of DNA damage signaling through the ATM-Chk2 pathway, which induces it to migrate to the nucleus during viral replication.IMPORTANCEThe chicken anemia virus (CAV) protein apoptin is known to induce tumor cell-specific death when expressed. Therefore, understanding its regulation and mechanism of action could provide new insights into tumor cell biology. We have determined that checkpoint kinase 1 and 2 signaling is important for apoptin regulation and is a likely feature of both tumor cells and host cells producing virus progeny. Inhibition of checkpoint signaling prevents apoptin toxicity in tumor cells and attenuates CAV replication, suggesting it may be a future target for antiviral therapy.

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Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks

The Journal of Cell Biology ◽

10.1083/jcb.200510015 ◽

2006 ◽

Vol 172 (6) ◽

pp. 823-834 ◽

Cited By ~ 326

Author(s):

Michael J. Kruhlak ◽

Arkady Celeste ◽

Graham Dellaire ◽

Oscar Fernandez-Capetillo ◽

Waltraud G. Müller ◽

...

Keyword(s):

Dna Damage ◽

Living Cells ◽

Double Strand Breaks ◽

Dna Integrity ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Limited Mobility ◽

Energy Filtering ◽

Dna Damage Signaling ◽

Cell Biol

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion (Pilch, D.R., O.A. Sedelnikova, C. Redon, A. Celeste, A. Nussenzweig, and W.M. Bonner. 2003. Biochem. Cell Biol. 81:123–129; Morrison, A.J., and X. Shen. 2005. Cell Cycle. 4:568–571; van Attikum, H., and S.M. Gasser. 2005. Nat. Rev. Mol. Cell. Biol. 6:757–765). The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated (ATM; Bakkenist, C.J., and M.B. Kastan. 2003. Nature. 421:499–506). However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30–40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate–dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.

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Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation

The Journal of Cell Biology ◽

10.1083/jcb.202106116 ◽

2021 ◽

Vol 220 (12) ◽

Author(s):

Delisa E. Clay ◽

Heidi S. Bretscher ◽

Erin A. Jezuit ◽

Korie B. Bush ◽

Donald T. Fox

Keyword(s):

Dna Damage ◽

Dna Polymerase ◽

Chromosome Segregation ◽

Genome Instability ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Dna Damage Signaling ◽

Alternative End Joining

Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophilamelanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.

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