In vivo γ-irradiation low dose threshold for suppression of DNA double strand breaks below the spontaneous level in mouse blood and spleen cells

ABSTRACT Orthologs of proteins SbcD (Mre11) and SbcC (Rad50) exist in all kingdoms of life and are involved in a wide variety of DNA repair and maintenance functions, including homologous recombination and nonhomologous end joining. Here, we have inactivated the sbcC and/or sbcD genes of Deinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced DNA double-strand breaks (DSB). Mutants devoid of the SbcC and/or SbcD proteins displayed reduced survival and presented a delay in kinetics of DSB repair and cell division following γ-irradiation. It has been recently reported that D. radiodurans DNA polymerase X (PolX) possesses a structure-modulated 3′-to-5′ exonuclease activity reminiscent of specific nuclease activities displayed by the SbcCD complex from Escherichia coli. We constructed a double mutant devoid of SbcCD and PolX proteins. The double-mutant ΔsbcCD ΔpolX Dr (where Dr indicates D. radiodurans) bacteria are much more sensitive to γ-irradiation than the single mutants, suggesting that the deinococcal SbcCD and PolX proteins may play important complementary roles in processing damaged DNA ends. We propose that they are part of a backup repair system acting to rescue cells containing DNA lesions that are excessively numerous or difficult to repair.

Download Full-text

Cellular responses to DNA double-strand breaks after low-dose γ-irradiation

Nucleic Acids Research ◽

10.1093/nar/gkp237 ◽

2009 ◽

Vol 37 (12) ◽

pp. 3912-3923 ◽

Cited By ~ 116

Author(s):

Aroumougame Asaithamby ◽

David J. Chen

Keyword(s):

Low Dose ◽

Cellular Responses ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Γ Irradiation ◽

Strand Breaks

Download Full-text

Fibroblasts from Ataxia Telangiectasia (AT) and AT Heterozygotes Show an Enhanced Level of Residual DNA Double-strand Breaks after Low Dose-rate γ-irradiation as Assayed by Pulsed Field Gel Electrophoresis

International Journal of Radiation Biology ◽

10.1080/09553009114552601 ◽

1991 ◽

Vol 60 (5) ◽

pp. 791-802 ◽

Cited By ~ 76

Author(s):

D. Blöcher ◽

D. Sigut ◽

M.A. Hannan

Keyword(s):

Dose Rate ◽

Ataxia Telangiectasia ◽

Gel Electrophoresis ◽

Low Dose ◽

Pulsed Field Gel Electrophoresis ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Γ Irradiation ◽

Strand Breaks ◽

Low Dose Rate

Download Full-text

Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo

DNA Repair ◽

10.1016/j.dnarep.2006.12.002 ◽

2007 ◽

Vol 6 (5) ◽

pp. 639-648 ◽

Cited By ~ 19

Author(s):

Yukitaka Katsura ◽

Shigeru Sasaki ◽

Masanori Sato ◽

Kiyoshi Yamaoka ◽

Kazumi Suzukawa ◽

...

Keyword(s):

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks

Download Full-text

Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

The Journal of Cell Biology ◽

10.1083/jcb.200608077 ◽

2007 ◽

Vol 177 (2) ◽

pp. 219-229 ◽

Cited By ~ 256

Author(s):

Naoya Uematsu ◽

Eric Weterings ◽

Ken-ichi Yano ◽

Keiko Morotomi-Yano ◽

Burkhard Jakob ◽

...

Keyword(s):

Kinase Activity ◽

Laser System ◽

Double Strand Breaks ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Dna Ends

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

Download Full-text

Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.00156-17 ◽

2017 ◽

Vol 37 (24) ◽

Cited By ~ 8

Author(s):

Sucheta Arora ◽

Rajashree A. Deshpande ◽

Martin Budd ◽

Judy Campbell ◽

America Revere ◽

...

Keyword(s):

Nuclease Activity ◽

Double Strand Breaks ◽

Endonuclease Activity ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Content Type ◽

Strand Breaks ◽

Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

Download Full-text

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

Cell Death and Differentiation ◽

10.1038/s41418-019-0406-6 ◽

2019 ◽

Vol 27 (4) ◽

pp. 1200-1213 ◽

Cited By ~ 1

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.

Download Full-text