Research Article

Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

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Science  01 May 2015:
Vol. 348, Issue 6234, 1253671
DOI: 10.1126/science.1253671
  • CHROMASS analysis of proteins recruited to stalled replication forks reveals a specific set of DNA repair factors involved in the replication stress response.

    Among these, SLF1 and SLF2 are found to bridge the SMC5/6 complex to RAD18, thereby linking SMC5/6 recruitment to ubiquitylation products formed at various DNA lesions.

  • Fig. 1 Dynamic recruitment of proteins to replication forks stalled at psoralen cross-links.

    Chromatin was replicated in Xenopus egg extract and analyzed by CHROMASS. (A) Analysis of protein recruitment to psoralen–cross-linked chromatin compared to an undamaged control. The volcano plot shows the mean difference of the protein intensity plotted against the P value. Dashed lines indicate the significance cutoff (16). (B) Protein recruitment to psoralen–cross-linked chromatin in the presence or absence of the replication inhibitor geminin. (C) Analysis of DNA damage checkpoint activation by probing total extracts with antibodies raised against phospho-CHK1. (D) CHROMASS analysis of chromatin pellets from the same reactions shown in (C). The heat map shows the relative abundance of the median intensity from three biological replicates calculated for each protein. See table S2 for intensities of all quantified proteins. A model for ICL repair is shown at the left. The same sample was analyzed in (A), (C), and (D).

  • Fig. 2 Pathway analysis reveals comprehensive proteomic coverage of the ICL repair pathway.

    (A) Time points evaluated for the identification of all damage-specific chromatin binders. Representative intensity profile of FancD2 on psoralen–cross-linked chromatin (red), undamaged chromatin (blue), or a mock control lacking chromatin (green) plotted against time. (B) The cumulative number of hits in each category plotted against the number of significant observations (table S1), in which a protein was found to be significantly enriched on psoralen–cross-linked chromatin relative to undamaged chromatin and a mock reaction. DDR, DNA damage response; COH, cohesion; REP, DNA replication; MISC, miscellaneous. See table S1 for assignment to categories. (C) Replication dependency of damage-specific chromatin binder with at least three significant observations. (D) Maximal intensity ratio (PSO/CTR) plotted against the rank of the protein. Dot and label sizes reflect the number of significant observations. (E) Mapping a global score for damage-specific enrichment (16) onto a schematic protein interaction network. Low coverage may indicate module members that are not involved in the response to cross-linking agents.

  • Fig. 3 SLF1 and SLF2 physically link RAD18 to the SMC5/6 complex.

    (A) Green fluorescent protein (GFP) pulldowns from HeLa (BAC) cells expressing SLF2, RAD18, or SMC6 as GFP fusion proteins under their endogenous promoter were analyzed by QUBIC (24). (B) Protein interactions identified in (A). Circle size indicates absolute copy number in HeLa cells (see fig. S7C). Baits are shown in dark orange. Arrow size indicates relative intensities of interactors. (C) U2OS cells left untreated or transfected with SLF1 siRNA were transfected with indicated FLAG-RAD18 constructs. Whole-cell extracts (WCE) were subjected to FLAG immunoprecipitation (IP) and immunoblotted with antibodies to SLF1, FLAG, and β-tubulin. (D) U2OS/FLAG-SLF2 cells were transfected with indicated SLF1 constructs. Interactions among SLF1, SLF2, and RAD18 were analyzed by immunoblotting GFP IPs with the indicated antibodies. (E) GFP IPs from HeLa or HeLa/GFP-RAD18 (BAC) cells transfected with indicated siRNAs were immunoblotted with antibody to SMC5. Knockdown efficiency of the SLF2 siRNA is shown in fig. S6F. (F) GFP IPs from U2OS cells transfected with the indicated siRNAs, followed by transfection with empty vector or GFP-SLF2 plasmid, were immunoblotted with antibodies to RAD18 and SMC5. (G) Schematic depiction of human SLF1 and SLF2 proteins. Conserved BRCT and ankyrin repeat (ANK) domains in SLF1 are highlighted. Interactions among RAD18, SLF1, SLF2, and SMC5/6 are indicated by double-headed arrows.

  • Fig. 4 The RAD18-SLF1-SLF2 complex promotes ubiquitin-dependent recruitment of SMC5/6 to sites of DNA damage.

    (A) U2OS/GFP-SLF1 cells transfected with indicated siRNAs were exposed to laser micro-irradiation, fixed 1 hour later, and immunostained with γ-H2AX antibody. Scale bar, 10 μm. (B and C) U2OS cells treated as in (A) were coimmunostained with antibody to SLF2 or SMC5, respectively. Scale bar, 10 μm. (D) Quantification of data shown in (A) to (C) and fig. S8A. At least 75 cells were counted per data point [mean ± SEM (error bars); N = 2]. (E) U2OS or U2OS/GFP-SLF1 cells were processed as in (B). At least 100 cells were counted per data point [mean ± SEM (error bars); N = 2]. See also fig. S8D. (F) CHROMASS analysis of protein recruitment to psoralen–cross-linked chromatin in the presence or absence of ubiquitin vinyl sulfone. (G) Protein recruitment to psoralen–cross-linked chromatin in the presence or absence of BRC4 peptide.

  • Fig. 5 Role of the RAD18-SLF1-SLF2 complex in genome stability maintenance after DNA damage.

    (A) Clonogenic survival of U2OS cells after exposure to ionizing radiation (mean ± SEM; N = 3). See also fig. S10A for an independent set of siRNAs. (B) Clonogenic survival of U2OS cells exposed to MMC (mean ± SEM; N = 3). (C) Chromosomal aberrations of U2OS cells exposed to MMC; 50 metaphases were analyzed per condition (mean ± SD; N = 2; ***P < 0.001, nonparametric t test, all tested against the siCTRL treated with MMC). (D) As in (E), except that either U2OS or U2OS/GFP-SLF1 cells were used (mean ± SD; N = 2; ***P < 0.001, *P < 0.05, nonparametric t test). (E and F) Proliferation of PSNG13 or PSNF5 cells (mean ± SD; N = 3). See also fig. S10D. (G) U2OS were exposed to ultraviolet (UV) irradiation. After 6 hours, extracts were analyzed by immunoblotting with antibodies to PCNA and MCM6. (H) U2OS/GFP-polη cells were treated as in (G) and analyzed by confocal microscopy. Scale bar, 10 μm. (I) Model of RAD18-SLF1-SLF2–mediated recruitment of the SMC5/6 complex to damaged DNA.

Supplementary Materials

  • Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

    Markus Räschle, Godelieve Smeenk, Rebecca K. Hansen, Tikira Temu, Yasuyoshi Oka, Marco Y. Hein, Nagarjuna Nagaraj, David T. Long, Johannes C. Walter, Kay Hofmann, Zuzana Storchova, Jürgen Cox, Simon Bekker-Jensen, Niels Mailand, Matthias Mann

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Supplementary Text
    • Materials and Methods
    • Captions for tables S1 and S2
    • Figs. S1 to S10
    • References
    Tables S1 and S2
    Table S1. Results from pairwise comparisons and classification. Summary of the results from pairwise comparisons using the SAM algorithm (22). For labelling and experimental conditions see Fig. S1B. Column B-BE report the test results (1: significant, 0: not significant, NA: not detected or not enough valid values). For all tests the significance threshold was an FDR<0.05 at a minimal fold change of 1.5. Column BT-EQ list the fold change and p-value (DIFF: difference of the LOG2 LFQ intensities; PVAL: –LOG10(p-value) of a standard Student's T-test). Volcano plots of each comparison are provided at the end of this supplement. For each time point of experiment EXP03-05 the subset of proteins with significant enrichment on psoralen crosslinked chromatin compared to a mock control was identified. This subset was further evaluated to identify proteins with a specific enrichment on psoralen crosslinked chromatin over the undamaged chromatin. Chromatin binders were defined as proteins with an NP Count > 0 (Column AH, sum of Column G,J,M,P,S,V,Y,AB,AE). Damage-specific chromatin binder (Column AK) were defined as proteins that showed a significant damage-specific enrichment over both controls in at least three conditions (Column AJ, sum of Column I,L,O,R,U,X,AA,AD,AG). Geminin-sensitive chromatin binder (Column F) were defined as proteins which showed at least once a geminin-sensitive enrichment on psoralen crosslinked chromatin (Column B-D). Evaluation of the RPC and pre-RC experiments are shown in Column AL-AO and AQ-AR, respectively (see Fig. S4 for details). For the damage-specific enrichment, a global score and maximum log2 fold change was calculated (reported in Column BF-BG) as described in Materials and Methods.

    Table S2. Dynamic recruitment of DNA repair factors to replication forks stalled at psoralen lesions. Relative abundance of the LFQ intensity of each quantified protein (column C-H). A subjective chronological order is provided in (column I). For each replicate the number of identified peptides (column J-AA) and the number of sequenced peptides (column AB-AS) are provided.

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