Article

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

ABSTRACT

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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    • "Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR) (Jackson and Durocher, 2013) [1]. USP3 is a histone H2A deubiquitinating enzyme (DUB) that regulates ubiquitin-dependent DDR in response to DNA double-strand breaks (Nicassio et al., 2007; Doil et al., 2008) [2] [3]. Deletion of USP3 in mice increases the incidence of spontaneous tumors and affects hematopoiesis [4]. "
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    ABSTRACT: The data described here provide genome-wide expression profiles of murine primitive hematopoietic stem and progenitor cells (LSK) and of B cell populations, obtained by high throughput sequencing. Cells are derived from wild-type mice and from mice deficient for the ubiquitin-specific protease 3 (USP3; Usp3Δ/Δ). Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR) (Jackson and Durocher, 2013) [1]. USP3 is a histone H2A deubiquitinating enzyme (DUB) that regulates ubiquitin-dependent DDR in response to DNA double-strand breaks (Nicassio et al., 2007; Doil et al., 2008) [2,3]. Deletion of USP3 in mice increases the incidence of spontaneous tumors and affects hematopoiesis [4]. In particular, Usp3-knockout mice show progressive loss of B and T cells and decreased functional potential of hematopoietic stem cells (HSCs) during aging. USP3-deficient cells, including HSCs, display enhanced histone ubiquitination, accumulate spontaneous DNA damage and are hypersensitive to ionizing radiation (Lancini et al., 2014) [4]. To address whether USP3 loss leads to deregulation of specific molecular pathways relevant to HSC homeostasis and/or B cell development, we have employed the RNA-sequencing technology and investigated transcriptional differences between wild-type and Usp3Δ/Δ LSK, naïve B cells or in vitro activated B cells. The data relate to the research article “Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells” (Lancini et al., 2014) [4]. The RNA-sequencing and analysis data sets have been deposited in NCBI׳s Gene Expression Omnibus (Edgar et al., 2002) [5] and are accessible through GEO Series accession number GSE58495 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58495). With this article, we present validation of the RNA-seq data set through quantitative real-time PCR and comparative analysis.
    Full-text · Article · Mar 2016
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    • "Although the structures for BRCA1 BRCT in complex with other phosphopeptides have been solved previously (Campbell et al., 2010; Clapperton et al., 2004; Liu and Ladias, 2013; Shen and Tong, 2008; Shiozaki et al., 2004; Varma et al., 2005; Williams et al., 2004), the structure for BRCT/Abraxas has remained unknown. Abraxas and the BRCA1-A complex recruit BRCA1 to DNA double-strand-break sites (DSBs) in an ATM-dependent ubiquitin-mediated signaling pathway involving E2 conjugase Ubc13, E3 ligases RNF8/RNF168, and Rap80 binding to ubiquitin lys63-linked polyubiquitin conjugates (Doil et al., 2009; Harper and Elledge, 2007; Hu et al., 2012; Huen and Chen, 2008; Kim et al., 2007b; Kolas et al., 2007; Lee and Paull, 2004; Mailand et al., 2007; Sato et al., 2009; Uziel et al., 2003; Wang, 2012; Wang and Elledge, 2007; Wu et al., 2009). Abraxas-deficient mice exhibit decreased survival and increased tumor incidence (Castillo et al., 2014). "
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    ABSTRACT: BRCA1 accumulation at DNA damage sites is an important step for its function in the DNA damage response and in DNA repair. BRCA1-BRCT domains bind to proteins containing the phosphorylated serine-proline-x-phenylalanine (pSPxF) motif including Abraxas, Bach1/FancJ, and CtIP. In this study, we demonstrate that ionizing radiation (IR)-induces ATM-dependent phosphorylation of serine 404 (S404) next to the pSPxF motif. Crystal structures of BRCT/Abraxas show that phosphorylation of S404 is important for extensive interactions through the N-terminal sequence outside the pSPxF motif and leads to formation of a stable dimer. Mutation of S404 leads to deficiency in BRCA1 accumulation at DNA damage sites and cellular sensitivity to IR. In addition, two germline mutations of BRCA1 are found to disrupt the dimer interface and dimer formation. Thus, we demonstrate a mechanism involving IR-induced phosphorylation and dimerization of the BRCT/Abraxas complex for regulating Abraxas-mediated recruitment of BRCA1 in response to IR.
    Full-text · Article · Jan 2016 · Molecular cell
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    • "H2AX at DSB sites is poly-ubiquitinated at K13 or K15, mediated by RNF168 in an MDC1-, NBS1-, and RNF8-dependent manner (van Attikum and Gasser, 2009). Together with RNF8, RNF168 catalyzes the formation of non-proteolytic K63-linked ubiquitin chains, which are involved in the recruitment of BRCA1 and 53BP1 (Doil et al., 2009; Mattiroli et al., 2012), in which 53BP1 directly recognizes ubiquitinated H2A/H2AX at the K15 site (Fradet-Turcotte et al., 2013). "
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    ABSTRACT: In response to DNA double-strand breaks (DSBs), H2AX is rapidly phosphorylated at Ser139 to promote DSB repair. Here we show that H2AX is rapidly stabilized in response to DSBs to efficiently generate γH2AX foci. This mechanism operated even in quiescent cells that barely expressed H2AX. H2AX stabilization resulted from the inhibition of proteasome-mediated degradation. Synthesized H2AX ordinarily underwent degradation through poly-ubiquitination mediated by the E3 ligase HUWE1; however, H2AX ubiquitination was transiently halted upon DSB formation. Such rapid H2AX stabilization by DSBs was associated with chromatin incorporation of H2AX and halting of its poly-ubiquitination mediated by the ATM kinase, the sirtuin protein SIRT6, and the chromatin remodeler SNF2H. H2AX Ser139, the ATM phosphorylation site, was essential for H2AX stabilization upon DSB formation. Our results reveal a pathway controlled by ATM, SIRT6, and SNF2H to block HUWE1, which stabilizes H2AX and induces its incorporation into chromatin only when cells are damaged.
    Full-text · Article · Dec 2015 · Cell Reports
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