HERC2 coordinates Ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes

Centre for Genotoxic Stress Research, Department of Proteomics in Cancer, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark.
Nature Cell Biology (Impact Factor: 19.68). 04/2010; 12(4):412. DOI: 10.1038/ncb0410-412
Source: PubMed


Regulatory ubiquitylation is emerging as an important mechanism to protect genome integrity in cells exposed to DNA damage1, 2, 3, 4, 5, 6, 7, 8, 9. However, the spectrum of known ubiquitin regulators of the DNA damage response (DDR) is limited and their functional interplay is poorly understood. Here, we identify HERC2 as a factor that regulates ubiquitin-dependent retention of repair proteins on damaged chromosomes. In response to ionising radiation (IR), HERC2 forms a complex with RNF8, a ubiquitin ligase involved in the DDR3, 4, 5, 6. The HERC2–RNF8 interaction requires IR-inducible phosphorylation of HERC2 at Thr 4827, which in turn binds to the forkhead-associated (FHA) domain of RNF8. Mechanistically, we provide evidence that HERC2 facilitates assembly of the ubiquitin-conjugating enzyme Ubc13 with RNF8, thereby promoting DNA damage-induced formation of Lys 63-linked ubiquitin chains. We also show that HERC2 interacts with, and maintains the levels of, RNF168, another ubiquitin ligase operating downstream of RNF8 (Refs 7, 8). Consequently, knockdown of HERC2 abrogates ubiquitin-dependent retention of repair factors such as 53BP1, RAP80 and BRCA1. Together with the increased radiosensitivity of HERC2-depleted cells, these results uncover a regulatory layer in the orchestration of protein interactions on damaged chromosomes and they underscore the role of ubiquitin-mediated signalling in genome maintenance.

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    • "Although these conjugates may serve distinct purposes during DNA damage-repair processes, the need to orchestrate these different outcomes requires multiple levels of regulation and fine-tuning. Indeed, the importance of achieving homeostatic balance of the dynamic ubiquitin environment is highlighted by the emergence of regulatory factors that innervate the ubiquitin-dependent DNA damage signaling cascade at the RNF8-RNF168 level (15,16,26–28,37–46). In particular, the ubiquitin-selective segregase VCP/p97 was recently found to promote DNA damage signaling by dismantling DSB-associated lys48-linked ubiquitin conjugates (15,16). "
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    ABSTRACT: Ubiquitylation plays key roles in DNA damage signal transduction. The current model envisions that lysine63-linked ubiquitin chains, via the concerted action of E3 ubiquitin ligases RNF8-RNF168, are built at DNA double-strand breaks (DSBs) to effectively assemble DNA damage-repair factors for proper checkpoint control and DNA repair. We found that RNF168 is a short-lived protein that is stabilized by the deubiquitylating enzyme USP34 in response to DNA damage. In the absence of USP34, RNF168 is rapidly degraded, resulting in attenuated DSB-associated ubiquitylation, defective recruitment of BRCA1 and 53BP1 and compromised cell survival after ionizing radiation. We propose that USP34 promotes a feed-forward loop to enforce ubiquitin signaling at DSBs and highlight critical roles of ubiquitin dynamics in genome stability maintenance.
    Full-text · Article · Jul 2013 · Nucleic Acids Research
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    • "Another HECT E3 ligase, HERC2, had been previously reported by the same group to have an opposite effect on RNF168 protein levels, by promoting RNF168 stability and therefore facilitating the assembly of repair factors to DSBs (Bekker-Jensen et al., 2010). In addition, the authors also showed that HERC2 was able to promote DSB signaling and repair by facilitating the assembly of RNF8 with its E2 partner UBC13, thus underscoring the importance of yet another E3 ligase, HERC2, in the RNF8/RNF168-dependent pathway in response to DSBs. "
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    ABSTRACT: RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligases comprise a large family of enzymes that in combination with an E2 ubiquitin-conjugating enzyme, modify target proteins by attaching ubiquitin moieties. A number of RING E3s play an essential role in the cellular response to DNA damage highlighting a crucial contribution for ubiquitin-mediated signaling to the genome surveillance pathway. Among the RING E3s, RNF8 and RNF168 play a critical role in the response to double stranded breaks, one of the most deleterious types of DNA damage. These proteins act as positive regulators of the signaling cascade that initiates at DNA lesions. Inactivation of these enzymes is sufficient to severely impair the ability of cells to respond to DNA damage. Given their central role in the pathway, several layers of regulation act at this nodal signaling point. Here we will summarize current knowledge on the roles of RNF8 and RNF168 in maintaining genome integrity with particular emphasis on recent insights into the multiple layers of regulation that act on these enzymes to fine-tune the cellular response to DNA lesions.
    Full-text · Article · Jul 2013 · Frontiers in Genetics
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    • "Interestingly, following the induction of DSBs both the ubiquitin and SUMO conjugation pathways function in concert to stimulate repair of damaged DNA. This ubiquitin/SUMO-dependent DDR (UbS-DDR) is mediated by an E3 ubiquitin/SUMO ligase cascade composed of RNF8, HERC2, RNF168, PIAS1 and PIAS4, which promotes alterations in the local chromatin architecture surrounding the break to allow the efficient recruitment of essential repair factors such as the BRCA1-A complex and 53BP1 (Kolas et al, 2007; Mailand et al, 2007; Wang et al, 2007; Doil et al, 2009; Galanty et al, 2009; Stewart et al, 2009; Bekker-Jensen et al, 2010; Mattiroli et al, 2012). Given the link between BLM cellular localization regulation and the SUMO system (Eladad et al, 2005; Ouyang et al, 2009), it is conceivable that ubiquitin may also play a key role in regulating BLM recruitment to sites of DNA damage. "
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    ABSTRACT: Limiting the levels of homologous recombination (HR) that occur at sites of DNA damage is a major role of BLM helicase. However, very little is known about the mechanisms dictating its relocalization to these sites. Here, we demonstrate that the ubiquitin/SUMO-dependent DNA damage response (UbS-DDR), controlled by the E3 ligases RNF8/RNF168, triggers BLM recruitment to sites of replication fork stalling via ubiquitylation in the N-terminal region of BLM and subsequent BLM binding to the ubiquitin-interacting motifs of RAP80. Furthermore, we show that this mechanism of BLM relocalization is essential for BLM's ability to suppress excessive/uncontrolled HR at stalled replication forks. Unexpectedly, we also uncovered a requirement for RNF8-dependent ubiquitylation of BLM and PML for maintaining the integrity of PML-associated nuclear bodies and as a consequence the localization of BLM to these structures. Lastly, we identified a novel role for RAP80 in preventing proteasomal degradation of BLM in unstressed cells. Taken together, these data highlight an important biochemical link between the UbS-DDR and BLM-dependent pathways involved in maintaining genome stability.
    Full-text · Article · May 2013 · The EMBO Journal
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