scholarly journals FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2126
Author(s):  
Lorenzo Lafranchi ◽  
Erik Müllers ◽  
Dorothea Rutishauser ◽  
Arne Lindqvist
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cyclin B1 ◽  
Live Cells ◽  
Dependent Manner ◽  
Mitotic Entry ◽  
Protein Levels ◽  
Partial Inhibition ◽  
Damage Response ◽  
G2 Phase

Cells recovering from the G2/M DNA damage checkpoint rely more on Aurora A-PLK1 signaling than cells progressing through an unperturbed G2 phase, but the reason for this discrepancy is not known. Here, we devised a method based on a FRET reporter for PLK1 activity to sort cells in distinct populations within G2 phase. We employed mass spectroscopy to characterize changes in protein levels through an unperturbed G2 phase and validated that ATAD2 levels decrease in a proteasome-dependent manner. Comparing unperturbed cells with cells recovering from DNA damage, we note that at similar PLK1 activities, recovering cells contain higher levels of Cyclin B1 and increased phosphorylation of CDK1 targets. The increased Cyclin B1 levels are due to continuous Cyclin B1 production during a DNA damage response and are sustained until mitosis. Whereas partial inhibition of PLK1 suppresses mitotic entry more efficiently when cells recover from a checkpoint, partial inhibition of CDK1 suppresses mitotic entry more efficiently in unperturbed cells. Our findings provide a resource for proteome changes during G2 phase, show that the mitotic entry network is rewired during a DNA damage response, and suggest that the bottleneck for mitotic entry shifts from CDK1 to PLK1 after DNA damage.

scholarly journals APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression

2019 ◽  
Author(s):  
Hiroyuki Yamazaki ◽  
Kotaro Shirakawa ◽  
Tadahiko Matsumoto ◽  
Yasuhiro Kazuma ◽  
Hiroyuki Matsui ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Dna Damage Response ◽  
Cytosine Deaminase ◽  
Myeloma Cell ◽  
Egfp Expression ◽  
Protein Levels ◽  
Dna Mutations ◽  
Damage Response ◽  
Editing Enzyme

AbstractApolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of A3B alone in cancer cells. To investigate A3B function in myeloma cells easily and comprehensively, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced under known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeting drugs, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed the EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage response triggers A3B expression through ATM, ATR and DNA-PK signaling.

scholarly journals ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation

2019 ◽  
Vol 150 (5) ◽  
pp. 1022-1030 ◽  
Author(s):  
Dandan Xu ◽  
Weiwei Dai ◽  
Lydia Kutzler ◽  
Holly A Lacko ◽  
Leonard S Jefferson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Damage ◽  
Amino Acid ◽  
Protein Kinase ◽  
Dna Damage Response ◽  
Dependent Manner ◽  
Mouse Embryo Fibroblasts ◽  
Damage Response ◽  
Mtorc1 Activation ◽  
In Cells

ABSTRACT Background The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. Objective The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. Methods Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0–16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. Results Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. Conclusions The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

scholarly journals Ribosomal protein L6 (RPL6) is recruited to DNA damage sites in a poly(ADP-ribose) polymerase–dependent manner and regulates the DNA damage response

2018 ◽  
Vol 294 (8) ◽  
pp. 2827-2838 ◽  
Author(s):  
Chuanzhen Yang ◽  
Weicheng Zang ◽  
Yapeng Ji ◽  
Tingting Li ◽  
Yongfeng Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ribosomal Protein ◽  
Dna Damage Response ◽  
Dependent Manner ◽  
Damage Response

Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 723-723
Author(s):  
Alexandra Sobeck ◽  
Stacie Stone ◽  
Bendert deGraaf ◽  
Vincenzo Costanzo ◽  
Johan deWinter ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
S Phase ◽  
Dependent Manner ◽  
Core Complex ◽  
Damage Response ◽  
Crosslinking Agents

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

scholarly journals ATM regulates a DNA damage response posttranscriptional RNA operon in lymphocytes

Blood ◽  
2011 ◽  
Vol 117 (8) ◽  
pp. 2441-2450 ◽  
Author(s):  
Krystyna Mazan-Mamczarz ◽  
Patrick R. Hagner ◽  
Yongqing Zhang ◽  
Bojie Dai ◽  
Elin Lehrmann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Ataxia Telangiectasia ◽  
Rna Binding ◽  
Critical Role ◽  
Cell Cycle Checkpoints ◽  
Dependent Manner ◽  
Damage Response ◽  
Multiple Cell

Abstract Maintenance of genomic stability depends on the DNA damage response, a biologic barrier in early stages of cancer development. Failure of this response results in genomic instability and high predisposition toward lymphoma, as seen in patients with ataxia-telangiectasia mutated (ATM) dysfunction. ATM activates multiple cell-cycle checkpoints and DNA repair after DNA damage, but its influence on posttranscriptional gene expression has not been examined on a global level. We show that ionizing radiation modulates the dynamic association of the RNA-binding protein HuR with target mRNAs in an ATM-dependent manner, potentially coordinating the genotoxic response as an RNA operon. Pharmacologic ATM inhibition and use of ATM-null cells revealed a critical role for ATM in this process. Numerous mRNAs encoding cancer-related proteins were differentially associated with HuR depending on the functional state of ATM, in turn affecting expression of encoded proteins. The findings presented here reveal a previously unidentified role of ATM in controlling gene expression posttranscriptionally. Dysregulation of this DNA damage response RNA operon is probably relevant to lymphoma development in ataxia-telangiectasia persons. These novel RNA regulatory modules and genetic networks provide critical insight into the function of ATM in oncogenesis.

scholarly journals Nuclear Accumulation of LAP1:TRF2 Complex during DNA Damage Response Uncovers a Novel Role for LAP1

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1804
Author(s):  
Cátia D. Pereira ◽  
Filipa Martins ◽  
Mariana Santos ◽  
Thorsten Müeller ◽  
Odete A. B. da Cruz e Silva ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cellular Response ◽  
Nuclear Accumulation ◽  
Protein Levels ◽  
Dna Damaging Agents ◽  
Physiological Properties ◽  
Damage Response

Lamina-associated polypeptide 1 (LAP1) is a nuclear envelope (NE) protein whose function remains poorly characterized. In a recent LAP1 protein interactome study, a putative regulatory role in the DNA damage response (DDR) has emerged and telomeric repeat-binding factor 2 (TRF2), a protein intimately associated with this signaling pathway, was among the list of LAP1 interactors. To gain insights into LAP1′s physiological properties, the interaction with TRF2 in human cells exposed to DNA-damaging agents was investigated. The direct LAP1:TRF2 binding was validated in vitro by blot overlay and in vivo by co-immunoprecipitation after hydrogen peroxide and bleomycin treatments. The regulation of this protein interaction by LAP1 phosphorylation was demonstrated by co-immunoprecipitation and mass spectrometry following okadaic acid exposure. The involvement of LAP1 and TRF2 in the DDR was confirmed by their increased nuclear protein levels after bleomycin treatment, evaluated by immunoblotting, as well as by their co-localization with DDR factors at the NE and within the nucleoplasm, assessed by immunocytochemistry. Effectively, we showed that the LAP1:TRF2 complex is established during a cellular response against DNA damage. This work proposes a novel functional role for LAP1 in the DDR, revealing a potential biological mechanism that may be disrupted in LAP1-associated pathologies.

scholarly journals Hof1 plays a checkpoint-related role in MMS-induced DNA damage response in Candida albicans

2020 ◽  
Vol 31 (5) ◽  
pp. 348-359 ◽  
Author(s):  
Jinrong Feng ◽  
Amjad Islam ◽  
Bjorn Bean ◽  
Jia Feng ◽  
Samantha Sparapani ◽  
...  
Keyword(s):  
Dna Damage ◽  
Candida Albicans ◽  
Mutant Strain ◽  
Dna Damage Response ◽  
Genotoxic Stress ◽  
Dependent Manner ◽  
Damage Response

Fifty-six strains from the GRACE collection were found to be sensitive to MMS upon repression. Deletion of the HOF1 gene renders sensitivity to genotoxic stress. Hof1 is genetically linked to the Rad53 pathway and is down-regulated in a Rad53-dependent manner. The importance of Hof1 in MMS response is reduced in a Rad23 or Rad4 mutant strain.

scholarly journals TFEB Modulates p21/WAF1/CIP1 during the DNA Damage Response

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1186 ◽  
Author(s):  
Sandra Pisonero-Vaquero ◽  
Chiara Soldati ◽  
Marcella Cesana ◽  
Andrea Ballabio ◽  
Diego Luis Medina
Keyword(s):  
Dna Damage ◽  
Cancer Cell ◽  
Dna Damage Response ◽  
Cell Types ◽  
Dependent Manner ◽  
Cancer Cell Growth ◽  
Direct Role ◽  
Growth And Survival ◽  
Damage Response ◽  
P21 Waf1

The MiT/TFE family of transcription factors (MITF, TFE3, and TFEB), which control transcriptional programs for autophagy and lysosome biogenesis have emerged as regulators of energy metabolism in cancer. Thus, their activation increases lysosomal catabolic function to sustain cancer cell growth and survival in stress conditions. Here, we found that TFEB depletion dramatically reduces basal expression levels of the cyclin-dependent kinase (CDK) inhibitor p21/WAF1 in various cell types. Conversely, TFEB overexpression increases p21 in a p53-dependent manner. Furthermore, induction of DNA damage using doxorubicin induces TFEB-mediated activation of p21, delays G2/M phase arrest, and promotes cell survival. Pharmacological inhibition of p21, instead, abrogates TFEB-mediated protection during the DNA damage response. Together, our findings uncover a novel and direct role of TFEB in the regulation of p21 expression in both steady-state conditions and during the induction of DNA-damage response (DDR). Our observations might open novel therapeutic strategies to promote cancer cell death by targeting the TFEB-p21 pathway in the presence of genotoxic agents.

scholarly journals Biphasic Functional Interaction between the Adenovirus E4orf4 Protein and DNA-PK

2019 ◽  
Vol 93 (10) ◽  
Author(s):  
Keren Nebenzahl-Sharon ◽  
Hassan Shalata ◽  
Rakefet Sharf ◽  
Jana Amer ◽  
Hanan Khoury-Haddad ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Virus Replication ◽  
Dna Damage Response ◽  
Late Phase ◽  
Direct Interaction ◽  
Dependent Manner ◽  
Functional Interaction ◽  
Cancer Cell Death ◽  
Damage Response

ABSTRACTThe adenovirus (Ad) E4orf4 protein contributes to virus-induced inhibition of the DNA damage response (DDR) by reducing ATM and ATR signaling. Consequently, E4orf4 inhibits DNA repair and sensitizes transformed cells to killing by DNA-damaging drugs. Inhibition of ATM and ATR signaling contributes to the efficiency of virus replication and may provide one explanation for the cancer selectivity of cell death induced by the expression of E4orf4 alone. In this report, we investigate a direct interaction of E4orf4 with the DDR. We show that E4orf4 physically associates with the DNA-dependent protein kinase (DNA-PK), and we demonstrate a biphasic functional interaction between these proteins, wherein DNA-PK is required for ATM and ATR inhibition by E4orf4 earlier during infection but is inhibited by E4orf4 as infection progresses. This biphasic process is accompanied by initial augmentation and a later inhibition of DNA-PK autophosphorylation as well as by colocalization of DNA-PK with early Ad replication centers and distancing of DNA-PK from late replication centers. Moreover, inhibition of DNA-PK improves Ad replication more effectively when a DNA-PK inhibitor is added later rather than earlier during infection. When expressed alone, E4orf4 is recruited to DNA damage sites in a DNA-PK-dependent manner. DNA-PK inhibition reduces the ability of E4orf4 to induce cancer cell death, likely because E4orf4 is prevented from arriving at the damage sites and from inhibiting the DDR. Our results support an important role for the E4orf4–DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.IMPORTANCESeveral DNA viruses evolved mechanisms to inhibit the cellular DNA damage response (DDR), which acts as an antiviral defense system. We present a novel mechanism by which the adenovirus (Ad) E4orf4 protein inhibits the DDR. E4orf4 interacts with the DNA damage sensor DNA-PK in a biphasic manner. Early during infection, E4orf4 requires DNA-PK activity to inhibit various branches of the DDR, whereas it later inhibits DNA-PK itself. Furthermore, although both E4orf4 and DNA-PK are recruited to virus replication centers (RCs), DNA-PK is later distanced from late-phase RCs. Delayed DNA-PK inhibition greatly contributes to Ad replication efficiency. When E4orf4 is expressed alone, it is recruited to DNA damage sites. Inhibition of DNA-PK prevents both recruitment and the previously reported ability of E4orf4 to kill cancer cells. Our results support an important role for the E4orf4–DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.

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