scholarly journals ATM-dependent DNA Damage-independent Mitotic Phosphorylation of H2AX in Normally Growing Mammalian Cells

2005 ◽  
Vol 16 (10) ◽  
pp. 5013-5025 ◽  
Author(s):  
Kirk J. McManus ◽  
Michael J. Hendzel
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Small Population ◽  
Histone H2a ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Mammalian Cell Lines ◽  
Carboxy Terminal

H2AX is a core histone H2A variant that contains an absolutely conserved serine/glutamine (SQ) motif within an extended carboxy-terminal tail. H2AX phosphorylation at the SQ motif (γ-H2AX) has been shown to increase dramatically upon exogenously introduced DNA double-strand breaks (DSBs). In this study, we use quantitative in situ approaches to investigate the spatial patterning and cell cycle dynamics of γ-H2AX in a panel of normally growing (unirradiated) mammalian cell lines and cultures. We provide the first evidence for the existence of two distinct yet highly discernible γ-H2AX focal populations: a small population of large amorphous foci that colocalize with numerous DNA DSB repair proteins and previously undescribed but much more abundant small foci. These small foci do not recruit proteins involved in DNA DSB repair. Cell cycle analyses reveal unexpected dynamics for γ-H2AX in unirradiated mammalian cells that include an ATM-dependent phosphorylation that is maximal during M phase. Based upon similarities drawn from other histone posttranslational modifications and previous observations in haploinsufficient (H2AX-/+) and null mice (H2AX-/-), γ-H2AX may contribute to the fidelity of the mitotic process, even in the absence of DNA damage, thereby ensuring the faithful transmission of genetic information from one generation to the next.

scholarly journals Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Songli Zhu ◽  
Mohammadjavad Paydar ◽  
Feifei Wang ◽  
Yanqiu Li ◽  
Ling Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

DNA double strand breaks (DSBs) have detrimental effects on cell survival and genomic stability, and are related to cancer and other human diseases. In this study, we identified microtubule-depolymerizing kinesin Kif2C as a protein associated with DSB-mimicking DNA templates and known DSB repair proteins in Xenopus egg extracts and mammalian cells. The recruitment of Kif2C to DNA damage sites was dependent on both PARP and ATM activities. Kif2C knockdown or knockout led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. Interestingly, Kif2C depletion, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Taken together, our study established Kif2C as a new player of the DNA damage response, and presented a new mechanism that governs DSB dynamics and repair.

scholarly journals Phosphorylation-Regulated Transitions in an Oligomeric State Control the Activity of the Sae2 DNA Repair Enzyme

2013 ◽  
Vol 34 (5) ◽  
pp. 778-793 ◽  
Author(s):  
Qiong Fu ◽  
Julia Chow ◽  
Kara A. Bernstein ◽  
Nodar Makharashvili ◽  
Sucheta Arora ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Posttranslational Modifications ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Oligomeric State ◽  
Content Type ◽  
Processing Enzymes

In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterizedin vivoin the budding yeastSaccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state.

scholarly journals Physical interaction between the histone acetyl transferase Tip60 and the DNA double-strand breaks sensor MRN complex

2010 ◽  
Vol 426 (3) ◽  
pp. 365-371 ◽  
Author(s):  
Catherine Chailleux ◽  
Sandrine Tyteca ◽  
Christophe Papin ◽  
François Boudsocq ◽  
Nadine Puget ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Histone Acetyl Transferase ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Dsbs

Chromatin modifications and chromatin-modifying enzymes are believed to play a major role in the process of DNA repair. The histone acetyl transferase Tip60 is physically recruited to DNA DSBs (double-strand breaks) where it mediates histone acetylation. In the present study, we show, using a reporter system in mammalian cells, that Tip60 expression is required for homology-driven repair, strongly suggesting that Tip60 participates in DNA DSB repair through homologous recombination. Moreover, Tip60 depletion inhibits the formation of Rad50 foci following ionizing radiation, indicating that Tip60 expression is necessary for the recruitment of the DNA damage sensor MRN (Mre11–Rad50–Nbs1) complex to DNA DSBs. Moreover, we found that endogenous Tip60 physically interacts with endogenous MRN proteins in a complex which is distinct from the classical Tip60 complex. Taken together, our results describe a physical link between a DNA damage sensor and a histone-modifying enzyme, and provide important new insights into the role and mechanism of action of Tip60 in the process of DNA DSB repair.

scholarly journals DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G0 cells

2021 ◽  
Author(s):  
Jessica K Tyler ◽  
Faith C Fowler ◽  
Chelsea Peart ◽  
Bo-Ruei Chen ◽  
Barry Sleckman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Proliferating Cells ◽  
Dsb Repair ◽  
Extensive Resection ◽  
Key Factor ◽  
Dna End Resection ◽  
End Resection

DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs are predominately repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in proliferating cells at the G1 or G2 phase of the cell cycle was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.

scholarly journals Phosphorylation of TIP60 Suppresses 53BP1 Localization at DNA Damage Sites

2018 ◽  
Vol 39 (1) ◽  
Author(s):  
Mischa Longyin Li ◽  
Qinqin Jiang ◽  
Natarajan V. Bhanu ◽  
Junmin Wu ◽  
Weihua Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Homologous Recombination ◽  
Posttranslational Modifications ◽  
Genome Stability ◽  
Parp Inhibitor ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Inhibitor Resistance

ABSTRACT A proper balance between the repair of DNA double-strand breaks (DSBs) by homologous recombination and nonhomologous end joining is critical for maintaining genome integrity and preventing tumorigenesis. This balance is regulated and fine-tuned by a variety of factors, including cell cycle and the chromatin environment. The histone acetyltransferase TIP60 was previously shown to suppress pathological end joining and promote homologous recombination. However, it is unknown how regulatory posttranslational modifications impact TIP60 acetyltransferase activity to influence the outcome of DSB responses. In this study, we report that phosphorylation of TIP60 on serines 90 and 86 is important for limiting the accumulation of the pro-end joining factor 53BP1 at DSBs in S and G2 cell cycle phases. Mutation of these sites disrupts histone acetylation changes in response to DNA damage, BRCA1 localization to DSBs, and poly(ADP-ribose) polymerase (PARP) inhibitor resistance. These findings reveal that phosphorylation directs TIP60-dependent acetylation to promote homologous recombination and maintain genome stability.

scholarly journals Actin-Related Protein 6 (Arp6) Influences Double-Strand Break Repair in Yeast

2021 ◽  
Vol 1 (2) ◽  
pp. 225-238
Author(s):  
Mohsen Hooshyar ◽  
Daniel Burnside ◽  
Maryam Hajikarimlou ◽  
Katayoun Omidi ◽  
Alexander Jesso ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Remodeling ◽  
Genetic Interaction ◽  
Interaction Analysis ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Dna Damaging Agents ◽  
Repair Efficiency ◽  
Chromosomal Breaks

DNA double-strand breaks (DSBs) are the most deleterious form of DNA damage and are repaired through non-homologous end-joining (NHEJ) or homologous recombination (HR). Repair initiation, regulation and communication with signaling pathways require several histone-modifying and chromatin-remodeling complexes. In budding yeast, this involves three primary complexes: INO80-C, which is primarily associated with HR, SWR1-C, which promotes NHEJ, and RSC-C, which is involved in both pathways as well as the general DNA damage response. Here we identify ARP6 as a factor involved in DSB repair through an RSC-C-related pathway. The loss of ARP6 significantly reduces the NHEJ repair efficiency of linearized plasmids with cohesive ends, impairs the repair of chromosomal breaks, and sensitizes cells to DNA-damaging agents. Genetic interaction analysis indicates that ARP6, MRE11 and RSC-C function within the same pathway, and the overexpression of ARP6 rescues rsc2∆ and mre11∆ sensitivity to DNA-damaging agents. Double mutants of ARP6, and members of the INO80 and SWR1 complexes, cause a significant reduction in repair efficiency, suggesting that ARP6 functions independently of SWR1-C and INO80-C. These findings support a novel role for ARP6 in DSB repair that is independent of the SWR1 chromatin remodeling complex, through an apparent RSC-C and MRE11-associated DNA repair pathway.

scholarly journals Dephosphorylation of the C-terminal Tyrosyl Residue of the DNA Damage-related Histone H2A.X Is Mediated by the Protein Phosphatase Eyes Absent

2009 ◽  
Vol 284 (24) ◽  
pp. 16066-16070 ◽  
Author(s):  
Navasona Krishnan ◽  
Dae Gwin Jeong ◽  
Suk-Kyeong Jung ◽  
Seong Eon Ryu ◽  
Andrew Xiao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Mammalian Cells ◽  
Protein Tyrosine Phosphatases ◽  
Histone H2a ◽  
Eyes Absent ◽  
Damage Repair ◽  
Damage Response

In mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of γH2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response.

scholarly journals Cooperative effects of genes controlling the G2/M checkpoint

2000 ◽  
Vol 14 (13) ◽  
pp. 1584-1588
Author(s):  
Timothy A. Chan ◽  
Paul M. Hwang ◽  
Heiko Hermeking ◽  
Kenneth W. Kinzler ◽  
Bert Vogelstein
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mammalian Cells ◽  
Growth Arrest ◽  
Double Knockout ◽  
Specific Test ◽  
Cooperative Effects ◽  
Exogenous Expression ◽  
Induced Genes ◽  
Single Knockout

It is believed that multiple effectors independently control the checkpoints permitting transitions between cell cycle phases. However, this has not been rigorously demonstrated in mammalian cells. The p53-induced genes p21 and 14-3-3ς are each required for the G2 arrest and allow a specific test of this fundamental tenet. We generated human cells deficient in bothp21 and 14-3-3ς and determined whether the double knockout was more sensitive to DNA damage than either single knockout.p21−/−14-3-3ς−/− cells were significantly more sensitive to DNA damage or to the exogenous expression of p53 than cells lacking only p21 or only 14-3-3ς. Thus, p21 and 14-3-3ς play distinct but complementary roles in the G2/M checkpoint, and help explain why genes at the nodal points of growth arrest pathways, like p53, are the targets of mutation in cancer cells.

scholarly journals The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

2020 ◽  
Author(s):  
Kenta Shinoda ◽  
Dali Zong ◽  
Elsa Callen ◽  
Wei Wu ◽  
Lavinia C. Dumitrache ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Instability ◽  
Parp Inhibitor ◽  
Progression Free Survival ◽  
Strand Break ◽  
Transcriptional Network ◽  
Cross Resistance ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Protein Levels

AbstractThe Shieldin complex, consisting of SHLD1, SHLD2, SHLD3 and REV7, shields DNA double strand breaks (DSBs) from nucleolytic resection. The end-protecting activity of Shieldin promotes productive non-homologous end joining (NHEJ) in G1 but can threaten genome integrity during S-phase by blocking homologous recombination (HR). Curiously, the penultimate Shieldin component, SHLD1 is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1 and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1. Functionally, this transcriptional network ensures that SHLD1 protein levels are kept in check to enable a proper balance between end protection and end resection during physiological DSB repair. In the context of BRCA1 deficiency, loss of THAP1 dependent SHLD1 expression confers cross resistance to PARP inhibitor and cisplatin, and shorter progression free survival in ovarian cancer patients. In contrast, loss of THAP1 in BRCA2 deficient cells increases genome instability and correlates with improved responses to chemotherapy. Pathogenic THAP1 mutations are causatively linked to the adult-onset torsion dystonia type 6 (DYT6) movement disorder, but the critical disease targets are unknown. We further demonstrate that murine models of Thap1-associated dystonia show reduced Shld1 expression concomitant with elevated levels of unresolved DNA damage in the brain. In summary, our study provides the first example of a transcriptional network that directly controls DSB repair choice and reveals a previously unsuspected link between DNA damage and dystonia.

scholarly journals Global Epigenetic Analysis Reveals H3K27 Methylation as a Mediator of Double Strand Break Repair

2021 ◽  
Author(s):  
Julian Lutze ◽  
Donald Wolfgeher ◽  
Stephen J. Kron
Keyword(s):  
Dna Damage ◽  
Cell Fate ◽  
Strand Break ◽  
Extensive Literature ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Checkpoint Signaling ◽  
H3k27 Methylation ◽  
Proteomic Data ◽  
Global Impacts

AbstractThe majority of cancer patients is treated with ionizing radiation (IR), a relatively safe and effective treatment considered to target tumors by inducing DNA double strand breaks (DSBs). Despite clinical interest in increasing the efficacy of IR by preventing successful DSB repair, few effective radio-adjuvant therapies exist. Extensive literature suggests that chromatin modifiers play a role in the DSB repair and thus may represent a novel class of radiosensitizers. Indeed, chromatin has both local and global impacts on DSB formation, recognition of breaks, checkpoint signaling, recruitment of repair factors, and timely DSB resolution, suggesting that epigenetic deregulation in cancer may impact the efficacy of radiotherapy. Here, using tandem mass spectrometry proteomics to analyze global patterns of histone modification in MCF7 breast cancer cells following IR exposure, we find significant and long-lasting changes to the epigenome. Our results confirm that H3K27 trimethylation (H3K27me3), best known for mediating gene repression and regulating cell fate, increases after IR. H3K27me3 changes rapidly, accumulating at sites of DNA damage. Inhibitors of the Polycomb related complex subunit and H3K27 methyltransferase EZH2 confirm that H3K27me3 is necessary for DNA damage recognition and cell survival after IR. These studies provide an argument for evaluating EZH2 as a radiosensitization target and H3K27me3 as a marker for radiation response in cancer. Proteomic data are available via ProteomeXchange with identifier PXD019388.

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