scholarly journals A DNA Damage-Regulated BRCT-Containing Protein, TopBP1, Is Required for Cell Survival

2002 ◽  
Vol 22 (2) ◽  
pp. 555-566 ◽  
Author(s):  
Kazuhiko Yamane ◽  
Xianglin Wu ◽  
Junjie Chen
Keyword(s):  
Dna Damage ◽  
Cell Survival ◽  
Ataxia Telangiectasia ◽  
Topoisomerase Ii ◽  
Sequence Similarity ◽  
Dna Double Strand Breaks ◽  
Focus Formation ◽  
Replication Checkpoint ◽  
Strand Breaks

ABSTRACT BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage-signaling pathways. Human DNA topoisomerase II binding protein 1 (TopBP1) contains eight BRCT motifs and shares sequence similarity with the fission yeast Rad4/Cut5 protein and the budding yeast DPB11 protein, both of which are required for DNA damage and/or replication checkpoint controls. We report here that TopBP1 is phosphorylated in response to DNA double-strand breaks and replication blocks. TopBP1 forms nuclear foci and localizes to the sites of DNA damage or the arrested replication forks. In response to DNA strand breaks, TopBP1 phosphorylation depends on the ataxia telangiectasia mutated protein (ATM) in vivo. However, ATM-dependent phosphorylation of TopBP1 does not appear to be required for focus formation following DNA damage. Instead, focus formation relies on one of the BRCT motifs, BRCT5, in TopBP1. Antisense Morpholino oligomers against TopBP1 greatly reduced TopBP1 expression in vivo. Similar to that of ataxia telangiectasia-related protein (ATR), Chk1, or Hus1, downregulation of TopBP1 leads to reduced cell survival, probably due to increased apoptosis. Taken together, the data presented here suggest that, like its putative counterparts in yeast species, TopBP1 may be involved in DNA damage and replication checkpoint controls.

scholarly journals DNA Damage-Induced Cell Cycle Regulation and Function of Novel Chk2 Phosphoresidues

2006 ◽  
Vol 26 (21) ◽  
pp. 7832-7845 ◽  
Author(s):  
Giacomo Buscemi ◽  
Luigi Carlessi ◽  
Laura Zannini ◽  
Sofia Lisanti ◽  
Enrico Fontanella ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Dna Double Strand Breaks ◽  
Independent Manner ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
And Function ◽  
Cycle Regulation

ABSTRACT Chk2 kinase is activated by DNA damage to regulate cell cycle arrest, DNA repair, and apoptosis. Phosphorylation of Chk2 in vivo by ataxia telangiectasia-mutated (ATM) on threonine 68 (T68) initiates a phosphorylation cascade that promotes the full activity of Chk2. We identified three serine residues (S19, S33, and S35) on Chk2 that became phosphorylated in vivo rapidly and exclusively in response to ionizing radiation (IR)-induced DNA double-strand breaks in an ATM- and Nbs1-dependent but ataxia telangiectasia- and Rad3-related-independent manner. Phosphorylation of these residues, restricted to the G1 phase of the cell cycle, was induced by a higher dose of IR (>1 Gy) than that required for phosphorylation of T68 (0.25 Gy) and declined by 45 to 90 min, concomitant with a rise in Chk2 autophosphorylation. Compared to the wild-type form, Chk2 with alanine substitutions at S19, S33, and S35 (Chk2S3A) showed impaired dimerization, defective auto- and trans-phosphorylation activities, and reduced ability to promote degradation of Hdmx, a phosphorylation target of Chk2 and regulator of p53 activity. Besides, Chk2S3A failed to inhibit cell growth and, in response to IR, to arrest G1/S progression. These findings underscore the critical roles of S19, S33, and S35 and argue that these phosphoresidues may serve to fine-tune the ATM-dependent response of Chk2 to increasing amounts of DNA damage.

scholarly journals DNA structure-specific priming of ATR activation by DNA-PKcs

2013 ◽  
Vol 202 (3) ◽  
pp. 421-429 ◽  
Author(s):  
Sophie Vidal-Eychenié ◽  
Chantal Décaillet ◽  
Jihane Basbous ◽  
Angelos Constantinou
Keyword(s):  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Protein A ◽  
Dna Structure ◽  
Specific Response ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Synergistic Activation ◽  
Dependent Protein ◽  
Specific Priming

Three phosphatidylinositol-3-kinase–related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)–covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.

scholarly journals βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1

2019 ◽  
Vol 27 (4) ◽  
pp. 1200-1213 ◽  
Author(s):  
Ainhoa Nieto ◽  
Makoto R. Hara ◽  
Victor Quereda ◽  
Wayne Grant ◽  
Vanessa Saunders ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Molecular Scaffold ◽  
Strand Breaks

Abstract Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal fidelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the β2-adrenergic-βarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, βarrestin-1 (βarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, MDM2. We sought to determine whether βarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice βarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that βarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of βarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, βarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, βarr1 is an important regulator of double strand break repair, and disruption of the βarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation.

scholarly journals Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks

2012 ◽  
Vol 40 (12) ◽  
pp. 5794-5794
Author(s):  
C. Lundin ◽  
M. North ◽  
K. Erixon ◽  
K. Walters ◽  
D. Jenssen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Methyl Methanesulfonate ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Heat Labile

scholarly journals Loss of p53 suppresses replication-stress-induced DNA breakage in G1/S checkpoint deficient cells

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Bente Benedict ◽  
Tanja van Harn ◽  
Marleen Dekker ◽  
Simone Hermsen ◽  
Asli Kucukosmanoglu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Replication Stress ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Origin Firing ◽  
Strand Breaks ◽  
Dna Breakage ◽  
Cycle Arrest

In cancer cells, loss of G1/S control is often accompanied by p53 pathway inactivation, the latter usually rationalized as a necessity for suppressing cell cycle arrest and apoptosis. However, we found an unanticipated effect of p53 loss in mouse and human G1-checkpoint-deficient cells: reduction of DNA damage. We show that abrogation of the G1/S-checkpoint allowed cells to enter S-phase under growth-restricting conditions at the expense of severe replication stress manifesting as decelerated DNA replication, reduced origin firing and accumulation of DNA double-strand breaks. In this system, loss of p53 allowed mitogen-independent proliferation, not by suppressing apoptosis, but rather by restoring origin firing and reducing DNA breakage. Loss of G1/S control also caused DNA damage and activation of p53 in an in vivo retinoblastoma model. Moreover, in a teratoma model, loss of p53 reduced DNA breakage. Thus, loss of p53 may promote growth of incipient cancer cells by reducing replication-stress-induced DNA damage.

ATM deficiency disrupts Tcra locus integrity and the maturation of CD4+CD8+ thymocytes

Blood ◽  
2006 ◽  
Vol 109 (5) ◽  
pp. 1887-1896 ◽  
Author(s):  
Irina R. Matei ◽  
Rebecca A. Gladdy ◽  
Lauryl M. J. Nutter ◽  
Angelo Canty ◽  
Cynthia J. Guidos ◽  
...  
Keyword(s):  
T Cell ◽  
Ataxia Telangiectasia ◽  
Cell Receptor ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Ataxia Telangiectasia Mutated ◽  
Apoptotic Pathway ◽  
Strand Breaks ◽  
Striking Contrast

Abstract Mutations in ATM (ataxia-telangiectasia mutated) cause ataxia-telangiectasia (AT), a disease characterized by neurodegeneration, sterility, immunodeficiency, and T-cell leukemia. Defective ATM-mediated DNA damage responses underlie many aspects of the AT syndrome, but the basis for the immune deficiency has not been defined. ATM associates with DNA double-strand breaks (DSBs), and some evidence suggests that ATM may regulate V(D)J recombination. However, it remains unclear how ATM loss compromises lymphocyte development in vivo. Here, we show that T-cell receptor β (TCRβ)–dependent proliferation and production of TCRβlow CD4+CD8+ (DP) thymocytes occurred normally in Atm−/− mice. In striking contrast, the postmitotic maturation of TCRβlow DP precursors into TCRβint DP cells and TCRβhi mature thymocytes was profoundly impaired. Furthermore, Atm−/− thymocytes expressed abnormally low amounts of TCRα mRNA and protein. These defects were not attributable to the induction of a BCL-2–sensitive apoptotic pathway. Rather, they were associated with frequent biallelic loss of distal Va gene segments in DP thymocytes, revealing that ATM maintains Tcra locus integrity as it undergoes V(D)J recombination. Collectively, our data demonstrate that ATM loss increases the frequency of aberrant Tcra deletion events, which compromise DP thymocyte maturation and likely promote the generation of oncogenic TCR translocations.

scholarly journals The BRCA2-Interacting Protein BCCIP Functions in RAD51 and BRCA2 Focus Formation and Homologous Recombinational Repair

2005 ◽  
Vol 25 (5) ◽  
pp. 1949-1957 ◽  
Author(s):  
Huimei Lu ◽  
Xu Guo ◽  
Xiangbing Meng ◽  
Jingmei Liu ◽  
Chris Allen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Stability ◽  
Recombinational Repair ◽  
Dna Double Strand Breaks ◽  
Focus Formation ◽  
Interacting Protein ◽  
Strand Breaks ◽  
Homologous Recombinational Repair ◽  
Radiation Induced ◽  
Nuclear Foci

ABSTRACT Homologous recombinational repair (HRR) of DNA damage is critical for maintaining genome stability and tumor suppression. RAD51 and BRCA2 colocalization in nuclear foci is a hallmark of HRR. BRCA2 has important roles in RAD51 focus formation and HRR of DNA double-strand breaks (DSBs). We previously reported that BCCIPα interacts with BRCA2. We show that a second isoform, BCCIPβ, also interacts with BRCA2 and that this interaction occurs in a region shared by BCCIPα and BCCIPβ. We further show that chromatin-bound BRCA2 colocalizes with BCCIP nuclear foci and that most radiation-induced RAD51 foci colocalize with BCCIP. Reducing BCCIPα by 90% or BCCIPβ by 50% by RNA interference markedly reduces RAD51 and BRCA2 foci and reduces HRR of DSBs by 20- to 100-fold. Similarly, reducing BRCA2 by 50% reduces RAD51 and BCCIP foci. These data indicate that BCCIP is critical for BRCA2- and RAD51-dependent responses to DNA damage and HRR.

scholarly journals Multiple autophosphorylation sites are dispensable for murine ATM activation in vivo

2008 ◽  
Vol 183 (5) ◽  
pp. 777-783 ◽  
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.

scholarly journals 53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response

2012 ◽  
Vol 197 (2) ◽  
pp. 283-300 ◽  
Author(s):  
Paula Martínez ◽  
Juana M. Flores ◽  
Maria A. Blasco
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Ataxia Telangiectasia ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Damage Response

TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)– and the ataxia telangiectasia and Rad3 related (ATR)–mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes.

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