Research ArticleDNA Repair

K63-linked polyubiquitin chains bind to DNA to facilitate DNA damage repair

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Science Signaling  05 Jun 2018:
Vol. 11, Issue 533, eaar8133
DOI: 10.1126/scisignal.aar8133
  • Fig. 1 K63-linked polyubiquitin chains interact with DNA in a chain length–dependent manner in vitro.

    (A to G) In vitro DNA binding assays for synthetic K63-linked (A), K48-linked (B), K11-linked (C), linearly linked (or M1-linked) (D), K29-linked (E), K33-linked (F), or K6-linked (G) ubiquitin chains. Shown are the subsequent immunoblotting (IB) analyses of biotin–double-stranded 70-mer (ds70mer) or biotin–single-stranded 70-nt (ss70nt) DNA pull-down assays with increasing amounts of synthetic ubiquitin chains. The topologies of ubiquitin chains (Ub3–7 or Ub4) with indicated linkages are presented on top of the blots. Green represents the Ile36 patch, and blue represents the Ile44 patch. Blots are representative of two experiments. (H and I) IB analyses of biotin-ds70mer or biotin-ss70nt DNA pull-down assays with indicated synthetic SUMOylation chains. Blots are representative of two experiments. (J) IB analyses of biotin-ds70mer or biotin-ss70nt DNA pull-down assays with indicated human influenza hemagglutinin (HA)–tagged synthetic monoubiquitin. Blots are representative of two experiments. (K) IB analyses of biotin-ds70mer DNA pull-down assays using each synthetic K63-linked ubiquitin chains with a fixed length as indicated. Blots are representative of two experiments. (L) IB analyses of gel filtration chromatography assays with synthetic tetra-ubiquitin chains of the indicated linkages in the presence or absence of ds70mer DNA. Assay used the Superdex 75 column in DNA binding buffers; conalbumin indicates the 75-kDa position, and ovalbumin (chicken) marks 44 kDa. Blots are representative of two experiments. (M) A summary table indicating that K63-linked chains of four or more ubiquitin molecules (n), but not other linkages of polyubiquitin chains examined, bind DNA in vitro. We could not examine whether K27-Ub4 chains bind DNA.

  • Fig. 2 K63-linked polyubiquitin chains interact with DNA through a DIP motif composed of adjacent Thr9, Lys11, and Glu34 residues.

    (A) IB analysis of ds30mer DNA pull-down assays from whole cell lysates (WCL) derived from human embryonic kidney (HEK) 293T cells transfected with HA-ubiquitin and treated with dimethyl sulfoxide (DMSO), etoposide (100 μM), doxorubicin (Dox; 10 μM), or cisplatin (20 μM) for 1 hour before harvesting and incubation with ds30mer DNA. Blots are representative of three experiments. (B to E) IB analyses of biotin-ds70mer DNA pull-down assays with WCLs derived from HEK293T cells that had been transfected with the indicated HA-tagged ubiquitin construct or empty vector (EV) control and treated with etoposide (20 μM) for 1 hour. Schematics of the structures of K63-only, K63/K11-only (K63/K11), and K63/K48-only (K63/K48) mutant polyubiquitin tested in (E) are presented in (D). Notably, EV represents empty vector as a negative control. Blots are representative of two experiments. WT, wild type. (F) Schematic of the location of residues spatially surrounding Lys11 derived from a crystal structure [Protein Data Bank (PDB: 3H7P)] of K63-diUb using the PyMOL Molecular Graphics System. Notably, the Lys11 residue is labeled in green to indicate a dual role of Lys11 in binding DNA and forming the K11 linkage, Thr9 and Glu34 are marked in red to illustrate their possible role involved in binding DNA, Thr12 and Thr14 are labeled in blue to indicate that they are not DIP residues, while the other “TEK” (threonine, glutamate, lysine) box residues including Leu8 and Ile36 are marked in yellow. (G and H) IB analyses of biotin-ds70mer DNA pull-down assays and WCLs derived from HEK293T cells transfected with indicated HA-ubiquitin constructs and treated with 20 μM etoposide for 1 hour before harvesting for the ds70mer DNA pull-down analysis. Blots are representative of two experiments. (I) Ubiquitin-DNA interaction model generated using a nuclear magnetic resonance solution structure of K63-linked di-ubiquitin (PDB: 2RR9) with DNA (PDB: 3BSE) using PyMOL.

  • Fig. 3 K63-linked polyubiquitin chains interact with DNA and Rap80 to form a ternary complex.

    (A) IB analysis of biotin-ds70mer DNA pull-downs in WCLs derived from HEK293 cells transfected with HA-ubiquitin and increasing amounts of Myc-His-Rap80 and, 48 hours later, treated with etoposide (20 μM) for 1 hour. Blots are representative of two experiments. (B) IB analysis of biotin-K63-Ub2-7 chain pull-down products with increasing doses of ds70mer DNA. Blots are representative of two experiments. GST, glutathione S-transferase. (C) IB of WCLs and HA–immunoprecipitations (IPs) derived from HEK293T cells transfected with indicated constructs and treated with etoposide (20 μM). Blot for HA in the WCL is shown in Fig. 2H. Blots are representative of two experiments. (D) Gel filtration analysis of biotin-ds70mer DNA incubated with K63-Ub4 in the presence or absence of bacterially purified GST-Rap80-2UIM protein at 4°C for 2 hours. (E) A computer modeling illustration for a putative complex composed of DNA, K63-linked di-ubiquitin, and the UIM domain of Rap80 using a DNA structure [PDB: 3BSE (55)] superimposed onto the solved structure of a K63-linked di-ubiquitin complexed with UIM domains of Rap80 (PDB: 3A1Q).

  • Fig. 4 The binding of DNA to K63-linked polyubiquitin chains is necessary for efficient DNA damage repair.

    (A and B) Sensitivity assays in yeast cells expressing the indicated WT or single or double mutant ubiquitin, cultured in YPD (yeast extract-peptone-dextrose) medium with either galactose (GALA) or glucose (GLU) and treated with MMS or etoposide. (C to E) Cell viability assays in human bone osteosarcoma epithelial U2OS cells transfected with the indicated ubiquitin construct that were cultured with doxycycline (1 μg/ml) for 36 hours and then treated with bleocin (C), etoposide (D), or doxorubicin (E) for another 36 hours. Data are means ± SD from three independent experiments. *P < 0.05 by Student’s t test. (F and G) Representative images (F) and quantification (G) of HA-tagged ubiquitin (green) colocalization with DNA damage (γH2Ax foci; red) upon laser stripping in U2OS cells in which endogenous ubiquitin had been knocked down and replaced with the indicated WT or mutant construct. Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 5 T9P or E34K ubiquitin mutants observed in cancer patients suppress the binding of K63-linked polyubiquitin chains to DNA.

    (A) Schematic of the patient tumor–derived ubiquitin mutations in UBC (UBC is a polyubiquitin gene in which E110K/E186K and T617P correspond to E34K and T9P in the ubiquitin protein), obtained from the cBioPortal database. Ub, monoubiquitin. (B) IB analysis of WCL and biotin-ds30mer DNA pull-downs derived from HEK293T cells transfected with the indicated construct of ubiquitin (EV) and Myc-tagged TRAF6. Blots are representative of two experiments. (C) Schematic of the proposed dominant-negative function of the mutated ubiquitin gene. If a mutant ubiquitin is present in any of four links of the ubiquitin chain, then it will lead to abolished DNA binding of the tetra-ubiquitin region. (D) IB analysis of WCL and biotin-ds30mer DNA pull-downs derived from HEK293T cells transfected with WT, HA-tagged ubiquitin (HA-Ub-WT), Myc-TRAF6, and the indicated additional WT or mutant ubiquitin construct. Blots are representative of two experiments. (E to G) Representative images (E) and quantification (F and G) of HCT116 cells stably expressing the indicated ubiquitin constructs exposed to 5-gray IR and recovered for 24 hours and then stained with antibodies against the indicated molecules. At least 100 cells were analyzed for each group. Data are means ± SD from three independent experiments. **P < 0.01 by Student’s t test. Scale bar, 10 μm.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/533/eaar8133/DC1

    Fig. S1. K63-linked polyubiquitin chains bind free and nucleosomal DNA in vitro.

    Fig. S2. K63-linked polyubiquitin chains bind DNA through a newly identified DIP motif including Thr9, Lys11, and Glu34 residues.

    Fig. S3. Binding to DNA does not disrupt but rather facilitate K63-linked polyubiquitin chains in binding proteins.

    Fig. S4. Deficiency in binding DNA leads to attenuated ability for K63-linked polyubiquitin chains in facilitating DDR.

    Fig. S5. Cancer patient–derived ubiquitin mutants in the DIP motif display attenuated ability in repairing damaged DNA in part due to deficiency in binding DNA.

    Table S1. Oligonucleotides.

    Table S2. Yeast strains used in this study.

    Reference (56)

  • Supplementary Materials for:

    K63-linked polyubiquitin chains bind to DNA to facilitate DNA damage repair

    Pengda Liu,* Wenjian Gan, Siyuan Su, Arthur V. Hauenstein, Tian-min Fu, Bradley Brasher, Carsten Schwerdtfeger, Anthony C. Liang, Ming Xu, Wenyi Wei*

    *Corresponding author. Email: pengda_liu{at}med.unc.edu (P.L.); wwei2{at}bidmc.harvard.edu (W.W.)

    This PDF file includes:

    • Fig. S1. K63-linked polyubiquitin chains bind free and nucleosomal DNA in vitro.
    • Fig. S2. K63-linked polyubiquitin chains bind DNA through a newly identified DIP motif including Thr9, Lys11, and Glu34 residues.
    • Fig. S3. Binding to DNA does not disrupt but rather facilitate K63-linked polyubiquitin chains in binding proteins.
    • Fig. S4. Deficiency in binding DNA leads to attenuated ability for K63-linked polyubiquitin chains in facilitating DDR.
    • Fig. S5. Cancer patient–derived ubiquitin mutants in the DIP motif display attenuated ability in repairing damaged DNA in part due to deficiency in binding DNA.
    • Table S1. Oligonucleotides.
    • Table S2. Yeast strains used in this study.
    • Reference (56)

    [Download PDF]


    © 2018 American Association for the Advancement of Science

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