RNF4-dependent hybrid SUMO-ubiquitin chains are signals for RAP80 and thereby mediate the recruitment of BRCA1 to sites of DNA damage

1Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA.
Science Signaling (Impact Factor: 6.28). 12/2012; 5(253):ra88. DOI: 10.1126/scisignal.2003485
Source: PubMed


The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double-strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. We demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin- and SUMO-dependent DSB recognition, revealing that RNF4-synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.

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    • "More recent work has revealed crucial roles for other types of ubiquitylation including monoubiquitylation and other types of polyubiquitin chain, in particular the K63 and K11 linkages (Ikeda & Dikic, 2008). Hybrid polyubiquitin chains made up of diverse ubiquitin linkages have also been detected in cells (Guzzo et al, 2012; Emmerich et al, 2013). The diverse polyubiquitin chains regulate cell functions such as gene transcription, DNA repair and replication, immune cell signalling, intracellular trafficking and many more (Hershko & Ciechanover, 1998; Kulathu & Komander, 2012). "
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    ABSTRACT: Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF-ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1-XPF. Together, these results establish USP45 as a new regulator of XPF-ERCC1 crucial for efficient DNA repair. © 2014 The Authors.
    The EMBO Journal 12/2014; 34(3). DOI:10.15252/embj.201489184 · 10.43 Impact Factor
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    • "In the case of arsenic trioxide, SUMO chains form on PML protein present in nuclear bodies, and RNF4 is rapidly recruited to these sites in a SIM-dependent, but RING-independent , fashion (Geoffroy et al., 2010). Similarly, DNA damage results in the accumulation of SUMO-2/3 chains at sites of DNA damage, again resulting in the recruitment of RNF4 to these localized nuclear subdomains (Galanty et al., 2012; Guzzo et al., 2012; Vyas et al., 2013; Yin et al., 2012). In contrast, heat stress or proteasome inhibition results in a global increase of SUMO chains (Golebiowski et al., 2009; Tatham et al., 2011). "
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    ABSTRACT: Dimeric RING E3 ligases interact with protein substrates and conformationally restrain the ubiquitin-E2-conjugating enzyme thioester complex such that it is primed for catalysis. RNF4 is an E3 ligase containing an N-terminal domain that binds its polySUMO substrates and a C-terminal RING domain responsible for dimerization. To investigate how RNF4 activity is controlled, we increased polySUMO substrate concentration by ablating expression of SUMO protease SENP6. Accumulation of SUMO chains in vivo leads to ubiquitin-mediated proteolysis of RNF4. In vitro we demonstrate that at concentrations equivalent to those found in vivo RNF4 is predominantly monomeric and inactive as an ubiquitin E3 ligase. However, in the presence of SUMO chains, RNF4 is activated by dimerization, leading to both substrate ubiquitylation and autoubiquitylation, responsible for degradation of RNF4. Thus the ubiquitin E3 ligase activity of RNF4 is directly linked to the availability of its polySUMO substrates.
    Molecular cell 03/2014; 53(6):880-92. DOI:10.1016/j.molcel.2014.02.031 · 14.02 Impact Factor
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    • "Many different E3 ubiquitin ligases participate in the DDR. For example, RNF2 catalyzes the monoubiquitination of H2AX contributing to ATM recruitment [32, 33], while depletion of RNF4 impairs RAP80, BRCA1, and RAD51 recruitment to sites of DNA damage [34–36]. "
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    International Journal of Cell Biology 09/2013; 2013(9):153634. DOI:10.1155/2013/153634
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