NBS1 is regulated by two kind of mechanisms: ATM-dependent complex formation with MRE11 and RAD50, and cell cycle–dependent degradation of protein

Abstract Nijmegen breakage syndrome (NBS), a condition similar to Ataxia-Telangiectasia (A-T), is a radiation-hypersensitive genetic disorder showing chromosomal instability, radio-resistant DNA synthesis, immunodeficiency, and predisposition to malignances. The product of the responsible gene, NBS1, forms a complex with MRE11 and RAD50 (MRN complex). The MRN complex is necessary for the DNA damage–induced activation of ATM. However, the regulation of MRN complex formation is still unclear. Here, we investigated the regulatory mechanisms of MRN complex formation. We used an immunoprecipitation assay to determine whether levels of the MRN complex were increased by radiation-induced DNA damage and found that the levels of these proteins and their mRNAs did not increase. ATM-dependent phosphorylation of NBS1 contributed to the DNA damage–induced MRN complex formation. However, pre-treatment of cells with an ATM-specific inhibitor did not affect homologous recombination (HR) and non-homologous end-joining (NHEJ) repair. G0 phase cells, decreasing NBS1 and HR activity but not NHEJ, gained HR-related chromatin association of RAD51 by overexpression of NBS1, suggesting that the amount of NBS1 may be important for repressing accidental activation of HR. These evidences suggest that NBS1 is regulated by two kind of mechanisms: complex formation dependent on ATM, and protein degradation mediated by an unknown MG132-resistant pathway. Such regulation of NBS1 may contribute to cellular responses to double-strand breaks.

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DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end-joining

Journal of Cell Science ◽

10.1242/jcs.249706 ◽

2021 ◽

pp. jcs.249706

Author(s):

Matteo Cabrini ◽

Marco Roncador ◽

Alessandro Galbiati ◽

Lina Cipolla ◽

Antonio Maffia ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Mrn Complex ◽

Break Site ◽

Non Homologous End Joining ◽

Dna Ends

The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSB) and promotes their resolution via the DNA repair pathways of Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). We and others have shown that DDR activation requires DROSHA. However, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment and how DROSHA influences DNA repair, remains poorly understood. Here we show that DROSHA associates to DSBs independently from transcription. Neither H2AX, nor ATM nor DNA-PK kinase activities are required for its recruitment to break site. Rather, DROSHA interacts with RAD50 and inhibition of MRN by Mirin treatment abolishes this interaction. MRN inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSB and, as a consequence, also 53BP1 recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increase the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and direct DNA repair toward NHEJ.

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Withanolide D Enhances Radiosensitivity of Human Cancer Cells by Inhibiting DNA Damage Non-homologous End Joining Repair Pathway

Frontiers in Oncology ◽

10.3389/fonc.2019.01468 ◽

2020 ◽

Vol 9 ◽

Author(s):

Jerome Lacombe ◽

Titouan Cretignier ◽

Laetitia Meli ◽

E. M. Kithsiri Wijeratne ◽

Jean-Luc Veuthey ◽

...

Keyword(s):

Dna Damage ◽

Cancer Cells ◽

Human Cancer ◽

Repair Pathway ◽

End Joining ◽

Human Cancer Cells ◽

Non Homologous End Joining

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The role of Schizosaccharomyces pombe Rad32, the Mre11 homologue, and other DNA damage response proteins in non-homologous end joining and telomere length maintenance

Nucleic Acids Research ◽

10.1093/nar/27.13.2655 ◽

1999 ◽

Vol 27 (13) ◽

pp. 2655-2661 ◽

Cited By ~ 50

Author(s):

S. Wilson ◽

N. Warr ◽

D. L. Taylor ◽

F. Z. Watts

Keyword(s):

Dna Damage ◽

Schizosaccharomyces Pombe ◽

Telomere Length ◽

Dna Damage Response ◽

End Joining ◽

Damage Response ◽

Non Homologous End Joining

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SIRT6 is a DNA double-strand break sensor

eLife ◽

10.7554/elife.51636 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 14

Author(s):

Lior Onn ◽

Miguel Portillo ◽

Stefan Ilic ◽

Gal Cleitman ◽

Daniel Stein ◽

...

Keyword(s):

Dna Damage ◽

Binding Capacity ◽

Critical Factor ◽

Strand Break ◽

Dna Double Strand Breaks ◽

End Joining ◽

Downstream Signaling ◽

The Road ◽

Dna Double Strand Break ◽

Non Homologous End Joining

DNA double-strand breaks (DSB) are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure that has high affinity for DSB. SIRT6 relocates to sites of damage independently of signaling and known sensors. It activates downstream signaling for DSB repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the homologous recombination and non-homologous end joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as a DNA damage sensor, a critical factor in initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB-binding capacity and DDR activation. SIRT6 activates the DDR before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 as a sensor of DSB, and pave the road to dissecting the contributions of distinct DSB sensors in downstream signaling.

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Proton Irradiation Increases the Necessity for Homologous Recombination Repair Along with the Indispensability of Non-Homologous End Joining

Cells ◽

10.3390/cells9040889 ◽

2020 ◽

Vol 9 (4) ◽

pp. 889 ◽

Cited By ~ 3

Author(s):

Klaudia Szymonowicz ◽

Adam Krysztofiak ◽

Jansje van der Linden ◽

Ajvar Kern ◽

Simon Deycmar ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Proton Irradiation ◽

Negative Impact ◽

Particle Therapy ◽

Homologous Recombination Repair ◽

End Joining ◽

X Ray ◽

Recombination Repair ◽

Non Homologous End Joining

Technical improvements in clinical radiotherapy for maximizing cytotoxicity to the tumor while limiting negative impact on co-irradiated healthy tissues include the increasing use of particle therapy (e.g., proton therapy) worldwide. Yet potential differences in the biology of DNA damage induction and repair between irradiation with X-ray photons and protons remain elusive. We compared the differences in DNA double strand break (DSB) repair and survival of cells compromised in non-homologous end joining (NHEJ), homologous recombination repair (HRR) or both, after irradiation with an equal dose of X-ray photons, entrance plateau (EP) protons, and mid spread-out Bragg peak (SOBP) protons. We used super-resolution microscopy to investigate potential differences in spatial distribution of DNA damage foci upon irradiation. While DNA damage foci were equally distributed throughout the nucleus after X-ray photon irradiation, we observed more clustered DNA damage foci upon proton irradiation. Furthermore, deficiency in essential NHEJ proteins delayed DNA repair kinetics and sensitized cells to both, X-ray photon and proton irradiation, whereas deficiency in HRR proteins sensitized cells only to proton irradiation. We assume that NHEJ is indispensable for processing DNA DSB independent of the irradiation source, whereas the importance of HRR rises with increasing energy of applied irradiation.

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A role for the p53 tumour suppressor in regulating the balance between homologous recombination and non-homologous end joining

Open Biology ◽

10.1098/rsob.160225 ◽

2016 ◽

Vol 6 (9) ◽

pp. 160225 ◽

Cited By ~ 16

Author(s):

Sylvie Moureau ◽

Janna Luessing ◽

Emma Christina Harte ◽

Muriel Voisin ◽

Noel Francis Lowndes

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Tumour Suppressor ◽

Cycle Phase ◽

Repair Pathway ◽

End Joining ◽

Dsb Repair ◽

Pathway Choice ◽

Non Homologous End Joining ◽

End Resection

Loss of p53, a transcription factor activated by cellular stress, is a frequent event in cancer. The role of p53 in tumour suppression is largely attributed to cell fate decisions. Here, we provide evidence supporting a novel role for p53 in the regulation of DNA double-strand break (DSB) repair pathway choice. 53BP1, another tumour suppressor, was initially identified as p53 Binding Protein 1, and has been shown to inhibit DNA end resection, thereby stimulating non-homologous end joining (NHEJ). Yet another tumour suppressor, BRCA1, reciprocally promotes end resection and homologous recombination (HR). Here, we show that in both human and mouse cells, the absence of p53 results in impaired 53BP1 focal recruitment to sites of DNA damage induced by ionizing radiation. This effect is largely independent of cell cycle phase and the extent of DNA damage. In p53-deficient cells, diminished localization of 53BP1 is accompanied by a reciprocal increase in BRCA1 recruitment to DSBs. Consistent with these findings, we demonstrate that DSB repair via NHEJ is abrogated, while repair via homology-directed repair (HDR) is stimulated. Overall, we propose that in addition to its role as an ‘effector’ protein in the DNA damage response, p53 plays a role in the regulation of DSB repair pathway choice.

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