RNF168 Binds and Amplifies Ubiquitin Conjugates on Damaged Chromosomes to Allow Accumulation of Repair Proteins

Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Copenhagen, Denmark.
Cell (Impact Factor: 32.24). 03/2009; 136(3):435-46. DOI: 10.1016/j.cell.2008.12.041
Source: PubMed


DNA double-strand breaks (DSBs) not only interrupt the genetic information, but also disrupt the chromatin structure, and both impairments require repair mechanisms to ensure genome integrity. We showed previously that RNF8-mediated chromatin ubiquitylation protects genome integrity by promoting the accumulation of repair factors at DSBs. Here, we provide evidence that, while RNF8 is necessary to trigger the DSB-associated ubiquitylations, it is not sufficient to sustain conjugated ubiquitin in this compartment. We identified RNF168 as a novel chromatin-associated ubiquitin ligase with an ability to bind ubiquitin. We show that RNF168 interacts with ubiquitylated H2A, assembles at DSBs in an RNF8-dependent manner, and, by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. Thus, RNF168 defines a new pathway involving sequential ubiquitylations on damaged chromosomes and uncovers a functional cooperation between E3 ligases in genome maintenance.

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Available from: Patrice Ménard, Nov 11, 2014
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    • "Covalent post-translational modification of proteins by ubiquitin and ubiquitin-like factors has emerged as a general mechanism to modulate DNA damage response (DDR) pathways (Jackson and Durocher, 2013; Jacq et al., 2013). Ubiquitin-based DSB signaling by RNF8 and RNF168 has been well established in DDR (Doil et al., 2009; Huen et al., 2007; Kolas et al., 2007; Lukas et al., 2011; Mailand et al., 2007; Mattiroli et al., 2012). So far, however, we only have a relatively limited understanding of the DDR roles for deubiquitylation enzymes (DUBs), which mediate the processing of DNA end resection. "
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    ABSTRACT: DNA end resection is a highly regulated and critical step in DNA double-stranded break (DSB) repair. In higher eukaryotes, DSB resection is initiated by the collaborative action of CtIP and the MRE11-RAD50-NBS1 (MRN) complex. Here, we find that the deubiquitylating enzyme USP4 directly participates in DSB resection and homologous recombination (HR). USP4 confers resistance to DNA damage-inducing agents. Mechanistically, USP4 interacts with CtIP and MRN via a specific, conserved region and the catalytic domain of USP4, respectively, and regulates CtIP recruitment to sites of DNA damage. We also find that USP4 autodeubiquitylation is essential for its HR functions. Collectively, our findings identify USP4 as a key regulator of DNA DSB end resection.
    Cell Reports 09/2015; 13(1). DOI:10.1016/j.celrep.2015.08.056 · 8.36 Impact Factor
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    • "eases ( USP ) USP3 H2A , H2AX , H2B , RIG1 Antagonizes RNF168 IRIFs ; promotes , DSB repair , sensitization : IR 1 Cell cycle ; type I interferon signaling ; HGF - dependent scattering response Usp3 - null mice display lymphopenia , decline in HSC function and spontaneous tumorigenesis upon aging Nicassio et al . ( 2007 ) , Buus et al . ( 2009 ) , Doil et al . ( 2009 ) , Mosbech et al . ( 2013 ) , Cui et al . ( 2014 ) , Lancini et al . ( 2014 ) , Nishi et al . ( 2014 ) , Sharma et al . ( 2014 ) USP16 / Ubp - M H2A 2 DSB - induced gene silencing Cell cycle ; transcription ; ESC differentiation Usp16 knockout is embryonic lethal . Trisomy of Usp16 ( Ts65Dn model for Down ' s syndrome ) associates with"
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    ABSTRACT: Ubiquitination is a reversible protein modification broadly implicated in cellular functions. Signaling processes mediated by ubiquitin are crucial for the cellular response to DNA double-strand breaks (DSBs), one of the most dangerous types of DNA lesions. In particular, the DSB response critically relies on active ubiquitination by the RNF8 and RNF168 ubiquitin ligases at the chromatin, which is essential for proper DSB signaling and repair. How this pathway is fine-tuned and what the functional consequences are of its deregulation for genome integrity and tissue homeostasis are subject of intense investigation. One important regulatory mechanism is by reversal of substrate ubiquitination through the activity of specific deubiquitinating enzymes (DUBs), as supported by the implication of a growing number of DUBs in DNA damage response (DDR) processes. Here, we discuss the current knowledge of how ubiquitin-mediated signaling at DSBs is controlled by deubiquitinating enzymes, with main focus on DUBs targeting histone H2A and on their recent implication in stem cell biology and cancer.
    Frontiers in Genetics 09/2015; 08(September 2015):Article 282. DOI:10.3389/fgene.2015.00282
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    • "Moreover, our results are consistent with previous studies showing that ethanol causes oxidative damage to DNA (Cherian et al. 2008; Chu et al. 2007; Lamarche et al. 2004; Rulten et al. 2008), which may generate DSBs. DNA repair by the NHEJ pathway includes the signaling of DNA damage sites, which involves the phosphorylation of the histone H2AX, ubiquitylation of the histone H2A by the E3 ubiquitin ligases RNF8 and RNF168, and recruitment of 53BP1 (Doil et al. 2009; Lieber 2010). The latter protects DNA ends from excessive resection by exonucleases and also orchestrates the aggregation of other repair factors required to restore genome integrity (Callen et al. 2013; Panier and Boulton 2014). "
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    ABSTRACT: Chronic alcohol consumption may cause neurodevelopmental and neurodegenerative disorders. Alcohol neurotoxicity is associated with the production of acetaldehyde and reactive oxygen species that induce oxidative DNA damage. However, the molecular mechanisms by which ethanol disturbs the DNA damage response (DDR), resulting in a defective DNA repair, remain unknown. Here, we have used cultured primary cortical neurons exposed to 50 or 100 mM ethanol for 7 days to analyze the ethanol-induced DDR. Ethanol exposure produced a dose-dependent generation of double strand breaks and the formation of DNA damage foci immunoreactive for the histone γH2AX, a DNA damage marker, and for the ubiquitylated H2A, which is involved in chromatin remodeling at DNA damage sites. Importantly, these DNA damage foci failed to recruit the protein 53BP1, a crucial DNA repair factor. This effect was associated with a drop in 53BP1 mRNA and protein levels and with an inhibition of global transcription. Moreover, ethanol-exposed neurons treated with ionizing radiation (2 Gy) also failed to recruit 53BP1 at DNA damage foci and exhibited a greater vulnerability to DNA lesions than irradiated control neurons. Our results support that defective DNA repair, mediated by the deficient expression and recruitment of 53BP1 to DNA damage sites, represents a novel mechanism involved in ethanol neurotoxicity. The design of therapeutic strategies that increase or stabilize 53BP1 levels might potentially promote DNA repair and partially compensate alcohol neurotoxicity.
    Neurotoxicity Research 08/2015; DOI:10.1007/s12640-015-9554-8 · 3.54 Impact Factor
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