scholarly journals 53BP1 Mediates ATR-Chk1 Signaling and Protects Replication Forks under Conditions of Replication Stress

2018 ◽  
Vol 38 (8) ◽  
Author(s):  
Joonyoung Her ◽  
Chandni Ray ◽  
Jake Altshuler ◽  
Haiyan Zheng ◽  
Samuel F. Bunting
Keyword(s):  
Cell Death ◽  
Cellular Response ◽  
Ex Vivo ◽  
Replication Stress ◽  
Strand Break ◽  
Replication Forks ◽  
P53 Signaling ◽  
Dna Double Strand Break ◽  
Short Term Exposure ◽  
Stress Signal

ABSTRACTComplete replication of the genome is an essential prerequisite for normal cell division, but a variety of factors can block the replisome, triggering replication stress and potentially causing mutation or cell death. The cellular response to replication stress involves recruitment of proteins to stabilize the replication fork and transmit a stress signal to pause the cell cycle and allow fork restart. We find that the ubiquitously expressed DNA damage response factor 53BP1 is required for the normal response to replication stress. Using primary,ex vivoB cells, we showed that a population of 53BP1−/−cells in early S phase is hypersensitive to short-term exposure to three different agents that induce replication stress. 53BP1 localizes to a subset of replication forks following induced replication stress, and an absence of 53BP1 leads to defective ATR-Chk1-p53 signaling and caspase 3-mediated cell death. Nascent replicated DNA additionally undergoes degradation in 53BP1−/−cells. These results show that 53BP1 plays an important role in protecting replication forks during the cellular response to replication stress, in addition to the previously characterized role of 53BP1 in DNA double-strand break repair.

scholarly journals DNA2 in Chromosome Stability and Cell Survival—Is It All about Replication Forks?

2021 ◽  
Vol 22 (8) ◽  
pp. 3984
Author(s):  
Jessica J. R. Hudson ◽  
Ulrich Rass
Keyword(s):  
Replication Stress ◽  
Strand Break ◽  
Replication Forks ◽  
Phenotypic Data ◽  
Checkpoint Activation ◽  
Dna Double Strand Break ◽  
Promising Target ◽  
Anti Cancer ◽  
Okazaki Fragment Processing ◽  
Response And Recovery

The conserved nuclease-helicase DNA2 has been linked to mitochondrial myopathy, Seckel syndrome, and cancer. Across species, the protein is indispensable for cell proliferation. On the molecular level, DNA2 has been implicated in DNA double-strand break (DSB) repair, checkpoint activation, Okazaki fragment processing (OFP), and telomere homeostasis. More recently, a critical contribution of DNA2 to the replication stress response and recovery of stalled DNA replication forks (RFs) has emerged. Here, we review the available functional and phenotypic data and propose that the major cellular defects associated with DNA2 dysfunction, and the links that exist with human disease, can be rationalized through the fundamental importance of DNA2-dependent RF recovery to genome duplication. Being a crucial player at stalled RFs, DNA2 is a promising target for anti-cancer therapy aimed at eliminating cancer cells by replication-stress overload.

scholarly journals Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination

2021 ◽  
Author(s):  
Kyosuke Nakamura ◽  
Georg Kustatscher ◽  
Constance Alabert ◽  
Martina Hödl ◽  
Ignasi Forne ◽  
...  
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Replication Forks ◽  
Dna Double Strand Break

scholarly journals DNA-PK: gatekeeper for IKKγ/NEMO nucleocytoplasmic shuttling in genotoxic stress-induced NF-kappaB activation

2020 ◽  
Vol 77 (20) ◽  
pp. 4133-4142
Author(s):  
Senad Medunjanin ◽  
Maximilian Putzier ◽  
Till Nöthen ◽  
Sönke Weinert ◽  
Thilo Kähne ◽  
...  
Keyword(s):  
Cellular Response ◽  
Phosphorylation Site ◽  
Genotoxic Stress ◽  
Nuclear Factor Κb ◽  
Strand Break ◽  
Nucleocytoplasmic Trafficking ◽  
Dependent Protein Kinase ◽  
Dna Double Strand Break ◽  
Dependent Protein ◽  
Nf Kappab

Abstract The transcription factors of the nuclear factor κB (NF-κB) family play a pivotal role in the cellular response to DNA damage. Genotoxic stress-induced activation of NF-κB differs from the classical canonical pathway by shuttling of the NF-κB Essential Modifier (IKKγ/NEMO) subunit through the nucleus. Here, we show that DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA double-strand break (DSB) repair, triggers the phosphorylation of NEMO by genotoxic stress, thereby enabling shuttling of NEMO through the nucleus with subsequent NF-κB activation. We identified serine 43 of NEMO as a DNA-PK phosphorylation site and point mutation of this serine to alanine led to a complete block of NF-κB activation by ionizing radiation (IR). Blockade of DNA-PK by a specific shRNA or by DNA-PKcs-deficient cells abrogated NEMO entry into the nucleus, as well. Accordingly, SUMOylation of NEMO, a prerequisite of nuclear NEMO, was abolished. Based on these observations, we propose a model in which NEMO phosphorylation by DNA-PK provides the first step in the nucleocytoplasmic trafficking of NEMO.

scholarly journals Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress

Cancers ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 355 ◽  
Author(s):  
Eric Moeglin ◽  
Dominique Desplancq ◽  
Sascha Conic ◽  
Mustapha Oulad-Abdelghani ◽  
Audrey Stoessel ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Cancer Cells ◽  
Drug Efficacy ◽  
Replication Stress ◽  
Strand Break ◽  
Histone H2ax ◽  
C Terminus ◽  
Dna Replication Stress ◽  
Stressed Cells

Phosphorylated histone H2AX (γ-H2AX), a central player in the DNA damage response (DDR), serves as a biomarker of DNA double-strand break repair. Although DNA damage is generally visualized by the formation of γ-H2AX foci in injured nuclei, it is unclear whether the widespread uniform nuclear γ-H2AX (called pan-nuclear) pattern occurring upon intense replication stress (RS) is linked to DDR. Using a novel monoclonal antibody that binds exclusively to the phosphorylated C-terminus of H2AX, we demonstrate that H2AX phosphorylation is systematically pan-nuclear in cancer cells stressed with RS-inducing drugs just before they die. The pan-nuclear γ-H2AX pattern is abolished by inhibition of the DNA-PK kinase. Cell death induction of cancer cells treated with increasing combinations of replication and kinase (ATR and Chk1) inhibitory drugs was proportional to the appearance of pan-nuclear γ-H2AX pattern. Delivery of labeled anti-γ-H2AX Fabs in stressed cells demonstrated at a single cell level that pan-nuclear γ-H2AX formation precedes irreversible cell death. Moreover, we show that H2AX is not required for RS-induced cell death in HeLa cells. Thus, the nuclear-wide formation of γ-H2AX is an incident of RS-induced cell death and, thus, the pan nuclear H2AX pattern should be regarded as an indicator of lethal RS-inducing drug efficacy.

scholarly journals Artemis-dependent DNA double-strand break formation at stalled replication forks

2013 ◽  
Vol 104 (6) ◽  
pp. 703-710 ◽  
Author(s):  
Junya Unno ◽  
Masatoshi Takagi ◽  
Jinhua Piao ◽  
Masataka Sugimoto ◽  
Fumiko Honda ◽  
...  
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Replication Forks ◽  
Dna Double Strand Break ◽  
Stalled Replication Forks ◽  
Break Formation

scholarly journals Beyond reversal: ubiquitin and ubiquitin-like proteases and the orchestration of the DNA double strand break repair response

2019 ◽  
Vol 47 (6) ◽  
pp. 1881-1893
Author(s):  
Alexander J. Garvin
Keyword(s):  
Protein Interactions ◽  
Cellular Response ◽  
Strand Break ◽  
Central Component ◽  
Protein Protein Interactions ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Post Translational Modifications ◽  
Strand Breaks ◽  
Dna Double Strand Break

The cellular response to genotoxic DNA double strand breaks (DSBs) uses a multitude of post-translational modifications to localise, modulate and ultimately clear DNA repair factors in a timely and accurate manner. Ubiquitination is well established as vital to the DSB response, with a carefully co-ordinated pathway of histone ubiquitination events being a central component of DSB signalling. Other ubiquitin-like modifiers (Ubl) including SUMO and NEDD8 have since been identified as playing important roles in DSB repair. In the last five years ∼20 additional Ub/Ubl proteases have been implicated in the DSB response. The number of proteases identified highlights the complexity of the Ub/Ubl signal present at DSBs. Ub/Ubl proteases regulate turnover, activity and protein–protein interactions of DSB repair factors both catalytically and non-catalytically. This not only ensures efficient repair of breaks but has a role in channelling repair into the correct DSB repair sub-pathways. Ultimately Ub/Ubl proteases have essential roles in maintaining genomic stability. Given that deficiencies in many Ub/Ubl proteases promotes sensitivity to DNA damaging chemotherapies, they could be attractive targets for cancer treatment.

scholarly journals Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

2014 ◽  
Vol 35 (5) ◽  
pp. 856-865 ◽  
Author(s):  
Arnab Ray Chaudhuri ◽  
Akshay Kumar Ahuja ◽  
Raquel Herrador ◽  
Massimo Lopes
Keyword(s):  
Cellular Response ◽  
Genome Stability ◽  
Microscopic Analysis ◽  
Strand Break ◽  
Replication Forks ◽  
Chromosomal Breakage ◽  
Electron Microscopic ◽  
Marked Accumulation ◽  
Chemotherapeutic Treatment ◽  
Replication Intermediates

Poly(ADP-ribosyl)ation (PAR) has been implicated in various aspects of the cellular response to DNA damage and genome stability. Although 17 human poly(ADP-ribose) polymerase (PARP) genes have been identified, a single poly(ADP-ribosyl) glycohydrolase (PARG) mediates PAR degradation. Here we investigated the role of PARG in the replication of human chromosomes. We show that PARG depletion affects cell proliferation and DNA synthesis, leading to replication-coupled H2AX phosphorylation. Furthermore, PARG depletion or inhibitionper seslows down individual replication forks similarly to mild chemotherapeutic treatment. Electron microscopic analysis of replication intermediates reveals marked accumulation of reversed forks and single-stranded DNA (ssDNA) gaps in unperturbed PARG-defective cells. Intriguingly, while we found no physical evidence for chromosomal breakage, PARG-defective cells displayed both ataxia-telangiectasia-mutated (ATM) and ataxia-Rad3-related (ATR) activation, as well as chromatin recruitment of standard double-strand-break-repair factors, such as 53BP1 and RAD51. Overall, these data prove PAR degradation to be essential to promote resumption of replication at endogenous and exogenous lesions, preventing idle recruitment of repair factors to remodeled replication forks. Furthermore, they suggest that fork remodeling and restarting are surprisingly frequent in unperturbed cells and provide a molecular rationale to explore PARG inhibition in cancer chemotherapy.

scholarly journals Cell fate determined by the activation balance between PKR and SPHK1

2020 ◽  
Vol 28 (1) ◽  
pp. 401-418
Author(s):  
Han Qiao ◽  
Tianqing Jiang ◽  
Peiqiang Mu ◽  
Xiaoxuan Chen ◽  
Xianhui Wen ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Fate ◽  
Cellular Response ◽  
Cellular Stress Response ◽  
Protein Kinase R ◽  
Survival Pathways ◽  
Stress Signal ◽  
Dependent Protein ◽  
Response To Stress ◽  
Simultaneous Activation

AbstractDouble-stranded RNA (dsRNA)-dependent protein kinase R (PKR) activation via autophosphorylation is the central cellular response to stress that promotes cell death or apoptosis. However, the key factors and mechanisms behind the simultaneous activation of pro-survival signaling pathways remain unknown. We have discovered a novel regulatory mechanism for the maintenance of cellular homeostasis that relies on the phosphorylation interplay between sphingosine kinase 1 (SPHK1) and PKR during exogenous stress. We identified SPHK1 as a previously unrecognized PKR substrate. Phosphorylated SPHK1, a central kinase, mediates the activation of PKR-induced pro-survival pathways by the S1P/S1PR1/MAPKs/IKKα signal axis, and antagonizes PKR-mediated endoplasmic reticulum (ER) stress signal transduction under stress conditions. Otherwise, phosphorylated SPHK1 also acts as the negative feedback factor, preferentially binding to the latent form of PKR at the C-terminal kinase motif, inhibiting the homodimerization of PKR, suppressing PKR autophosphorylation, and reducing the signaling strength for cell death and apoptosis. Our results suggest that the balance of the activation levels between PKR and SPHK1, a probable hallmark of homeostasis maintenance, determines cell fate during cellular stress response.

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