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 SET8 localization to chromatin flanking DNA damage is dependent on RNF168 ubiquitin ligase

2019 ◽  
Author(s):  
Stanimir Dulev ◽  
Sichun Lin ◽  
Qingquan Liu ◽  
Vildan B. Cetintas ◽  
Nizar N. Batada
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Proteasome Inhibition ◽  
Double Strand Breaks ◽  
Signaling Proteins ◽  
Dna Double Strand Breaks ◽  
Post Translational Modifications ◽  
Chromatin Remodelers ◽  
Strand Breaks

AbstractThe DNA damage response (DDR) associated post-translational modifications recruit chromatin remodelers, signaling proteins such as 53BP1 and repair factors to chromatin flanking DNA double strand breaks (DSBs) to promote its repair. Although localization of both RNF168 ubiquitin ligase and SET8 methyltransferase at DSBs is essential for 53BP1’s recruitment to DSBs, it is unclear if they do so via the same pathways. Here we report that RNF168 mediates SET8’s recruitment to DSBs. Depletion of cellular pool of ubiquitin through proteasome inhibition abolished RNF168 and SET8’s localization to DNA damage. Knockdown of RNF8 or RNF168 abolished SET8’s recruitment to DNA damage. Moreover, RNF168 and SET8 form stable complexes in vivo. Based on these results we propose a model in which SET8, which despite being a pan-chromatin binding protein, can accumulate several folds at chromatin flanking DSBs through tethering to other proteins that specifically localize to chromatin regions with specific modifications.

scholarly journals ATM-dependent pathways of chromatin remodelling and oxidative DNA damage responses

2017 ◽  
Vol 372 (1731) ◽  
pp. 20160283 ◽  
Author(s):  
N. Daniel Berger ◽  
Fintan K. T. Stanley ◽  
Shaun Moore ◽  
Aaron A. Goodarzi
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Strand Break ◽  
Chromatin Remodelling ◽  
Double Strand Breaks ◽  
Biochemical Network ◽  
Regulatory Function ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
The Impact

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase with a master regulatory function in the DNA damage response. In this role, ATM commands a complex biochemical network that signals the presence of oxidative DNA damage, including the dangerous DNA double-strand break, and facilitates subsequent repair. Here, we review the current state of knowledge regarding ATM-dependent chromatin remodelling and epigenomic alterations that are required to maintain genomic integrity in the presence of DNA double-strand breaks and/or oxidative stress. We will focus particularly on the roles of ATM in adjusting nucleosome spacing at sites of unresolved DNA double-strand breaks within complex chromatin environments, and the impact of ATM on preserving the health of cells within the mammalian central nervous system. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

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 SIRT6 is a DNA Double-Strand Break Sensor

2019 ◽  
Author(s):  
Lior Onn ◽  
Miguel Portillo ◽  
Stefan Ilic ◽  
Gal Cleitman ◽  
Daniel Stein ◽  
...  
Keyword(s):  
Dna Damage ◽  
Binding Capacity ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
The Road ◽  
Non Homologous End Joining

AbstractDNA double strand breaks are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure, with high affinity for double strand breaks. It relocates to sites of damage independently of signalling and known sensors and activates downstream signalling cascades for double strand break 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 DNA damage sensor, which is critical for 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 at the top of the DDR and pave the road to dissect the contributions of distinct double strand break sensors in downstream signalling.

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.

scholarly journals Tex19.1 Regulates Meiotic DNA Double Strand Break Frequency in Mouse Spermatocytes

2017 ◽  
Author(s):  
James H. Crichton ◽  
Christopher J. Playfoot ◽  
Marie MacLennan ◽  
David Read ◽  
Howard J. Cooke ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Meiotic Recombination ◽  
Homologous Chromosome ◽  
E3 Ubiquitin Ligase ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Genetic Pathway ◽  
Strand Breaks ◽  
Chromosome Synapsis ◽  
Meiotic Dsbs

AbstractMeiosis relies on the SPO11 endonuclease to generate the recombinogenic DNA double strand breaks (DSBs) required for homologous chromosome synapsis and segregation. The number of meiotic DSBs needs to be sufficient to allow chromosomes to search for and find their homologs, but not excessive to the point of causing genome instability. Here we report that meiotic DSB frequency in mouse spermatocytes is regulated by the mammal-specific gene Tex19.1. We show that the chromosome asynapsis previously reported in Tex19.1-/- spermatocytes is preceded by reduced numbers of recombination foci in leptotene and zygotene. Tex19.1 is required for the generation of normal levels of Spo11-dependent DNA damage during leptotene, but not for upstream events such as MEI4 foci formation or accumulation of H3K4me3 at recombination hotspots. Furthermore, we show that mice carrying mutations in the E3 ubiquitin ligase UBR2, a TEX19.1-interacting partner, phenocopy the Tex19.1-/- recombination defects. These data show that Tex19.1 and Ubr2 are required for mouse spermatocytes to generate sufficient meiotic DSBs to ensure that homology search is consistently successful, and reveal a hitherto unknown genetic pathway regulating meiotic DSB frequency in mammals.Author SummaryMeiosis is a specialised type of cell division that occurs during sperm and egg development to reduce chromosome number prior to fertilisation. Recombination is a key step in meiosis as it facilitates the pairing of homologous chromosomes prior to their reductional division, and generates new combinations of genetic alleles for transmission in the next generation. Regulating the amount of recombination is key for successful meiosis: too much will likely cause mutations, chromosomal re-arrangements and genetic instability, whereas too little causes defects in homologous chromosome pairing prior to the meiotic divisions. This study identifies a genetic pathway requiredto generate robust meiotic recombination in mouse spermatocytes. We show that male mice with mutations in Tex19.1 or Ubr2, which encodes an E3 ubiquitin ligase that interacts with TEX19.1, have defects in generating normal levels of meiotic recombination. We show that the defects in these mutants impact on the recombination process at the stage when programmed DNA double strand breaks are being made. This defect likely contributes to the chromosome synapsis and meiotic progression phenotypes previously described in these mutant mice. This study has implications for our understanding of how this fundamental aspect of genetics and inheritance is controlled.

scholarly journals A Role for DEAD Box 1 at DNA Double-Strand Breaks

2008 ◽  
Vol 28 (20) ◽  
pp. 6413-6425 ◽  
Author(s):  
Lei Li ◽  
Elizabeth A. Monckton ◽  
Roseline Godbout
Keyword(s):  
Ionizing Radiation ◽  
Neuroblastoma Cell ◽  
Rnase H ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Rna Helicases ◽  
Strand Breaks ◽  
Dead Box

ABSTRACT DEAD box proteins are a family of putative RNA helicases associated with all aspects of cellular metabolism involving the modification of RNA secondary structure. DDX1 is a member of the DEAD box protein family that is overexpressed in a subset of retinoblastoma and neuroblastoma cell lines and tumors. DDX1 is found primarily in the nucleus, where it forms two to four large aggregates called DDX1 bodies. Here, we report a rapid redistribution of DDX1 in cells exposed to ionizing radiation, resulting in the formation of numerous foci that colocalize with γ-H2AX and phosphorylated ATM foci at sites of DNA double-strand breaks (DSBs). The formation of DDX1 ionizing-radiation-induced foci (IRIF) is dependent on ATM, which was shown to phosphorylate DDX1 both in vitro and in vivo. The treatment of cells with RNase H prevented the formation of DDX1 IRIF, suggesting that DDX1 is recruited to sites of DNA damage containing RNA-DNA structures. We have shown that DDX1 has RNase activity toward single-stranded RNA, as well as ADP-dependent RNA-DNA- and RNA-RNA-unwinding activities. We propose that DDX1 plays an RNA clearance role at DSB sites, thereby facilitating the template-guided repair of transcriptionally active regions of the genome.

scholarly journals Megabase Chromatin Domains Involved in DNA Double-Strand Breaks in Vivo

1999 ◽  
Vol 146 (5) ◽  
pp. 905-916 ◽  
Author(s):  
Emmy P. Rogakou ◽  
Chye Boon ◽  
Christophe Redon ◽  
William M. Bonner
Keyword(s):  
Ionizing Radiation ◽  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Dna Integrity ◽  
Dna Double Strand Breaks ◽  
Unique Region ◽  
Strand Breaks ◽  
Histone H2ax ◽  
Visual Confirmation

The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named γ-H2AX. An antibody prepared to the unique region of human γ-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that γ-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, γ-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, γ-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that γ-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.

scholarly journals Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1

2019 ◽  
Author(s):  
Guillaume Gaullier ◽  
Genevieve Roberts ◽  
Uma M. Muthurajan ◽  
Samuel Bowerman ◽  
Johannes Rudolph ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Genome Instability ◽  
Structural Information ◽  
Regulatory Protein ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response

AbstractPoly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are relevant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleosomes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1•PARP2•nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.

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