RAD18 transmits DNA damage signaling to elicit homologous recombination repair

Department of Therapeutic Radiology, Yale University School of Medicine, P.O. Box 208040, New Haven, CT 06520, USA.
Nature Cell Biology (Impact Factor: 20.06). 05/2009; 11(5):592-603. DOI: 10.1038/ncb1865
Source: PubMed

ABSTRACT To maintain genome stability, cells respond to DNA damage by activating signalling pathways that govern cell-cycle checkpoints and initiate DNA repair. Cell-cycle checkpoint controls should connect with DNA repair processes, however, exactly how such coordination occurs in vivo is largely unknown. Here we describe a new role for the E3 ligase RAD18 as the integral component in translating the damage response signal to orchestrate homologous recombination repair (HRR). We show that RAD18 promotes homologous recombination in a manner strictly dependent on its ability to be recruited to sites of DNA breaks and that this recruitment relies on a well-defined DNA damage signalling pathway mediated by another E3 ligase, RNF8. We further demonstrate that RAD18 functions as an adaptor to facilitate homologous recombination through direct interaction with the recombinase RAD51C. Together, our data uncovers RAD18 as a key factor that orchestrates HRR through surveillance of the DNA damage signal.

  • Source
    • "RNF168 further induces protein ubiquitination events at DNA damage sites (Doil et al., 2009; Stewart et al., 2009). Moreover, HERC2, an E3 ligase partner of RNF8, and the downstream E3 ligase RAD18 also relocate to DNA damage sites and amplify the local ubiquitination events (Bekker-Jensen et al., 2010; Huang et al., 2009). These ubiquitination events are finally recognized by ubiquitin binding proteins, such as RAP80, which stabilizes other DNA damage response factors including BRCA1 and 53BP1 at DNA damage sites (Kim et al., 2007; Sobhian et al., 2007; Wang et al., 2007; Wu et al., 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similar to RNF168-deficient cells, RAD6A or 6B-deficient cells exhibit a reduction in DNA damage-induced protein ubiquitination. Correspondingly, DNA damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or 6B. Moreover, the RNF168/RAD6 complex targeted histone H1.2 for ubiquitination in vitro, and regulated DNA damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or 6B, is activated in the DNA damage-induced protein ubiquitination cascade.
    Journal of Cell Science 03/2013; 126(9). DOI:10.1242/jcs.122945 · 5.33 Impact Factor
  • Source
    • "Consequently, we found that the distribution of GFP-UBC13 and conjugated ubiquitin along DSB-containing chromatin was also significantly impaired after PARP inhibition (Fig. 3C,D). Ubiquitin conjugates generated by RNF8 and RNF168 mediate recruitment of ubiquitin-binding factors RAD18 and the RAP80– BRCA1 complex (Huang et al., 2009; Kim et al., 2007; Sobhian et al., 2007; Wang et al., 2007). Consistent with the defective spreading of ubiquitin conjugates, we found that PARPi also significantly reduced the expansion of GFP-RAD18, GFP-RAP80 and endogenous BRCA1 (Fig. 3C,D). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of γH2AX into damaged chromatin, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA damage. In support of this, we show that SMARCA5 and RNF168 interact in a DNA damage- and PARP-dependent manner. RNF168 becomes poly(ADP-ribosyl)ated after DNA damage, while RNF168 and poly(ADP-ribose) chains were required for SMARCA5 binding in vivo, explaining how SMARCA5 is linked to the RNF168 ubiquitin cascade. Moreover, SMARCA5 regulates the ubiquitin response by promoting RNF168 accumulation at DSBs, which subsequently facilitates efficient ubiquitin conjugation and BRCA1 assembly. Underlining the importance of these findings, we show that SMARCA5 depletion renders cells sensitive to IR and results in DSB repair defects. Our study unveils a functional link between DNA damage-induced poly(ADP-ribosyl)ation, SMARCA5-mediated chromatin remodeling and RNF168-dependent signaling and repair of DSBs.
    Journal of Cell Science 12/2012; 126(4). DOI:10.1242/jcs.109413 · 5.33 Impact Factor
  • Source
    • "The accumulation of RAD51, the recombinase required for DSB repair by HR, also requires RNF8, and is related to the extraction activity of p97/VCP but is independent of RNF168- generated K63-Ub chains (Stewart et al, 2007; Meerang et al, 2011; Sy et al, 2011). The post-replication repair Ub ligase RAD18 may also contribute by interacting both with RAD51C and with K63-poly-Ub or K48-poly-Ub generated by RNF8 (Huang et al, 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The regulation of Ubiquitin (Ub) conjugates generated by the complex network of proteins that promote the mammalian DNA double-strand break (DSB) response is not fully understood. We show here that the Ub protease POH1/rpn11/PSMD14 resident in the 19S proteasome regulatory particle is required for processing poly-Ub formed in the DSB response. Proteasome activity is required to restrict tudor domain-dependent 53BP1 accumulation at sites of DNA damage. This occurs both through antagonism of RNF8/RNF168-mediated lysine 63-linked poly-Ub and through the promotion of JMJD2A retention on chromatin. Consistent with this role POH1 acts in opposition to RNF8/RNF168 to modulate end-joining DNA repair. Additionally, POH1 acts independently of 53BP1 in homologous recombination repair to promote RAD51 loading. Accordingly, POH1-deficient cells are sensitive to DNA damaging agents. These data demonstrate that proteasomal POH1 is a key de-ubiquitinating enzyme that regulates ubiquitin conjugates generated in response to damage and that several aspects of the DSB response are regulated by the proteasome.
    The EMBO Journal 08/2012; 31(19):3918-34. DOI:10.1038/emboj.2012.232 · 10.75 Impact Factor