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. 2012 Dec 4;5(253):ra88.
doi: 10.1126/scisignal.2003485.

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

Catherine M Guzzo  1 , Christopher E Berndsen, Jianmei Zhu, Vibhor Gupta, Ajit Datta, Roger A Greenberg, Cynthia Wolberger, Michael J Matunis
Affiliations

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

Catherine M Guzzo et al. Sci Signal. .

Abstract

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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Conflict of interest statement

Competing interests: The authors have no competing interests to declare.

Figures

Fig. 1
Fig. 1
RAP80 contains a consensus SIM critical for recruitment to DSBs. (A) Diagram illustrating predicted ubiquitin-binding (blue) and SUMO-binding (orange) domains in components of the BRCA1-A complex. (SIM) SUMO-interacting motif; (UIM) ubiquitin-interacting motif; (AIR) Abraxas-interacting region; (ZnF) zinc fingers; (MPN±) MPR1/Pad1 N-terminal region ± active protease site; (CC) coiled-coil; (UEV1) ubiquitin E2 variant. (B) GST or GST-tagged SUMO-2(x3) was immobilized on glutathione-coated plates and incubated with the indicated FLAG- or His-tagged proteins. Bound proteins were eluted and analyzed by immunoblotting. Data shown are representative of XX experiments. (C) Diagrams of wild-type and mutant RAP80 N-terminal domains and RAP80 SIM. The UIM mutant is missing the second of the two UIMs. (D) GST or GST-tagged wild-type and mutant RAP80 N-terminal domains were immobilized on glutathione-coated plates and incubated with SUMO-2(x3) (left panel) or K63-Ub4 (right panel). Bound proteins were eluted and analyzed by immunoblotting with SUMO-, ubiquitin-, or GST-specific antibodies. Data shown are representative of XX experiments. (E) U2OS cells were transfected with constructs encoding full-length, GFP-tagged wild-type (WT), UIM, SIM, or UIM and SIM (UIM/SIM) mutants of RAP80. Cells were irradiated and recruitment to DNA repair foci was analyzed by fluorescence microscopy following a 2 h recovery. Bar = 5 μm. (F) Quantitative analysis of RAP80 recruitment to DNA repair foci following IR. Results are representative of three independent experiments. Error bars represent standard deviations.
Fig. 2
Fig. 2
RAP80 binds hybrid SUMO-ubiquitin chains with high affinity. (A) Schematic outline of the synthesis of hybrid SUMO-ubiquitin chains. RNF4, Ube2w and Ubc13-Mms2 represent E2 and E3 enzymes used in each step of synthesis. (B) GST-tagged RAP80 N-terminal domain was immobilized on glutathione-coated plates and incubated with SUMO-2, K63-linked diubiquitin, or hybrid SUMO-2~diubiquitin chains. Bound proteins were eluted and analyzed by immunoblotting with SUMO-2-, ubiquitin-, or GST-specific antibodies. Data shown are representative of XX experiments. (C) Similar binding assays similar to those described in B were performed with the wild-type RAP80 N-terminus and UIM or SIM mutants. (D) Quantitative analysis of in vitro binding to hybrid SUMO-2~diubiquitin chains shown in C. Results are representative of three independent experiments. Error bars represent standard deviations. (E) Quantitative binding results based on analytical ultracentifugation analysis of RAP80 N-terminal domain in complex with SUMO-2, K63-linked diubiquitin, or hybrid SUMO-2~diubiquitin chains.
Fig. 3
Fig. 3
RNF4 is recruited to DSBs and required for recruitment of RAP80 to DSBs. (A) Control and IR-treated U2OS cells were analyzed by immunofluorescence microscopy with antibodies specific for RNF4 or γH2AX. Bar = 5 μm. (B) U2OS cells were transfected with control or RNF4-specific siRNAs and subsequently treated with IR. Cells were allowed to recover for 2 h and recruitment of RAP80 to γH2AX-labeled repair foci was analyzed by immunofluorescence microscopy. Bar = 5 μm. (C) Quantitative analysis of RAP80 recruitment to γH2AX-labeled repair foci in cells transfected with control or RNF4-specific siRNAs. Data are presented as the percent of cells with more than 10 foci that were positive for both proteins. Results are representative of three independent experiments. Error bars equal standard deviations. (D) Quantitative analysis of RAP80 recruitment to γH2AX-labeled repair foci in cells cotransfected with RNF4-specific siRNA and RNAi-resistant RNF4-GFP cDNA. Analysis was performed as described in C.
Fig 4
Fig 4
RNF4 is required for the recruitment of BRCA1 to DSBs. (A) U2OS cells were transfected with control or RNF4-specific siRNAs and subsequently treated with IR. Cells were allowed to recover for 2 h and recruitment of BRCA1 to γH2AX-labeled repair foci was analyzed by immunofluorescence microscopy. Bar = 5 μm. (B) Quantitative analysis of BRCA1 recruitment to γH2AX-labeled repair foci in cells transfected with control or RNF4-specific siRNAs. Analyses were performed 0.5, 1.0, and 2.0 h after treatment with IR. Results are representative of three independent experiments. Error bars equal standard deviations. (C) Quantitative analysis of BRCA1 recruitment to γH2AX-labeled repair foci in cells cotransfected with RNF4-specific siRNA and RNAi-resistant RNF4-GFP cDNA. Analysis was performed 0.5 h after treatment with IR. (D) Model for hybrid SUMO-ubiquitin chain-dependent recruitment of RAP80 and the BRCA1-A complex to DSBs.
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