The E3 ligase RNF8 regulates Ku80 removal and NHEJ repair

Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 02/2012; 19(2):201-6. DOI: 10.1038/nsmb.2211
Source: PubMed


The ubiquitination cascade has a key role in the assembly of repair and signaling proteins at sites of double-strand DNA breaks. The E3 ubiquitin ligase RING finger protein 8 (RNF8) triggers the initial ubiquitination at double-strand DNA breaks, whereas sustained ubiquitination requires the downstream E3 ligase RING finger protein 168 (RNF168). It is not known whether RNF8 and RNF168 have discrete substrates and/or form different ubiquitin chains. Here we show that RNF168 acts with the ubiquitin-conjugating enzyme E2 13 (UBC13) and specifically synthesizes Lys63-linked chains, whereas RNF8 primarily forms Lys48-linked chains on chromatin, which promote substrate degradation. We also find that RNF8 regulates the abundance of the nonhomologous end-joining (NHEJ) repair protein KU80 at sites of DNA damage, and that RNF8 depletion results in prolonged retention of KU80 at damage sites and impaired nonhomologous end-joining repair. These findings reveal a distinct feature of RNF8 and indicate the involvement of the ubiquitination-mediated degradation pathway in DNA damage repair.

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    • "Inaddition,RNF8alsoubiquitylatesK48-dependentsub- stratessuchasthelysinedemethylaseJMJD2A(Malletteetal., 2012),theNHEJrepairproteinKu80(FengandChen,2012), andtheDNApolymeraseslidingclampproliferatingcellnuclear antigen(PCNA),whichisinvolvedinDNAsynthesisandrepair (Zhangetal.,2008).Consequently,theseproteinsareremoved fromchromatinforproteasomaldegradation. "
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    ABSTRACT: In response to DNA damage, cells activate a highly conserved and complex kinase-based signaling network, commonly referred to as the DNA damage response (DDR), to safeguard genomic integrity. The DDR consists of a set of tightly regulated events, including detection of DNA damage, accumulation of DNA repair factors at the site of damage, and finally physical repair of the lesion. Upon overwhelming damage the DDR provokes detrimental cellular actions by involving the apoptotic machinery and inducing a coordinated demise of the damaged cells (DNA damage-induced apoptosis, DDIA). These diverse actions involve transcriptional activation of several genes that govern the DDR. Moreover, recent observations highlighted the role of ubiquitylation in orchestrating the DDR, providing a dynamic cellular regulatory circuit helping to guarantee genomic stability and cellular homeostasis (Popovic et al., 2014). One of the hallmarks of human cancer is genomic instability (Hanahan and Weinberg, 2011). Not surprisingly, deregulation of the DDR can lead to human diseases, including cancer, and can induce resistance to genotoxic anti-cancer therapy (Lord and Ashworth, 2012). Here, we summarize the role of ubiquitin-signaling in the DDR with special emphasis on its role in cancer and highlight the therapeutic value of the ubiquitin-conjugation machinery as a target in anti-cancer treatment strategy.
    Full-text · Article · Apr 2015 · Frontiers in Genetics
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    • "Indeed, when DSBs occur in the S-phase, ubiquitination of two Fanconi Anemia proteins, FANCD2 and FANCI, promotes their accumulation to IRIFs [74] [75]. On the contrary , RNF8 triggers the ubiquitination-mediated Ku80 removal from damaged sites for an efficient NHEJ repair [76]. Moreover, other protein modifications take place at DSB, like small ubiquitin-like modifier (SUMO) on several DDR proteins [77]. "
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    ABSTRACT: The occurrence of DNA double-strand breaks (DSBs) induced by ionizing radiation has been extensively studied by biochemical or cell imaging techniques. Cell imaging development relies on technical advances as well as our knowledge of the cell DNA damage response (DDR) process. The DDR involves a complex network of proteins that initiate and coordinate DNA damage signaling and repair activities. As some DDR proteins assemble at DSBs in an established spatio-temporal pattern, visible nuclear foci are produced. In addition, post-translational modifications are important for the signaling and the recruitment of specific partners at damaged chromatin foci. We briefly review here the most widely used methods to study DSBs. We also discuss the development of indirect methods, using reporter expression or intra-nuclear antibodies, to follow the production of DSBs in real time and in living cells.
    Full-text · Article · Jul 2013 · Radiotherapy and Oncology
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    • "In agreement with the well characterized observation that RNF8 promotes the synthesis of K63-linked ubiquitin chains at DNA damage sites in somatic cells 9, K63-linked ubiquitination was enriched in the XY body and was absent in Rnf8 knockout spermatocytes (Figure 1b). Recent studies have shown that RNF8 also facilitates the transient assembly of K48-linked ubiquitin chains at DNA damage sites in somatic cells 10,11. Interestingly, K48-linked ubiquitination was also enriched in the XY body of wild type, but not Rnf8 knockout spermatocytes (Figure 1c). "
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    ABSTRACT: During meiotic prophase in males, the sex chromosomes partially synapse to form the XY body, a unique structure that recruits proteins involved in the DNA damage response, which is believed to be important for silencing of the sex chromosomes. It remains elusive how the DNA damage response in the XY body is regulated. Here we show that H2AX-MDC1-RNF8 signaling, which is well characterized in somatic cells, is dispensable for the recruitment of proteins to the unsynapsed axes in the XY body. On the other hand, the DNA damage response that spreads over the sex chromosomes is largely similar to that in somatic cells. This analysis shows that there are important differences between the regulation of the DNA damage response at the XY body and at DNA damage sites in somatic cells.
    Preview · Article · Jun 2013 · Nature Communications
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