Nature Structural and Molecular Biology: doi: /nsmb.2937
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2 Supplementary Figure 1 Multiple sequence alignment of the CtIP N-terminal domain, purified CtIP protein constructs and details of the 2F o F c electron density map of CtIP-NTD. (a) Multiple sequence alignment, including the predicted secondary structure (red rods indicate regions of predicted α-helix) and coiledcoil regions; the probability of coiled-coil formation is indicated by - (<50%), c (50-90%) or C (>90%). Protein sequence analysis was performed with the Jalview suite of bioinformatics tools ( Hs: Homo sapiens, Mm: Mus musculus, Ec: Equus caballus, Oc: Oryctolagus cuniculus, Gg: Gallus gallus, Xl: Xenopus laevis, Dr: Danio rerio. (b) SDS-PAGE analysis of purified recombinant CtIP protein samples: NTD, NTD-DM (C89A, C92A), nntd, cntd, cntd-dm (C89A, C92A) and CTD visualised by Coomassie staining. (c) Details of the 2F o - F c electron density map, contoured at 1.2 rmsd, superimposed on the refined crystallographic model of CtIP-NTD. The figure shows the map in the vicinity of amino acid W24. The map is shown as thin mesh in light purple, the protein atoms are shown in ball-and-stick representation and coloured according to chemical identity. The figure was prepared in Coot
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4 Supplementary Figure 2 CD analysis of CtIP N-terminal-domain constructs and micropixe analysis of the CtIP N-terminal domain. Circular dichroism (CD) analysis of CtIP N-terminal domain constructs. (a, c, e) Far-UV CD spectra recorded between 260 and 185 nm, in mean residue ellipticity, MRE ([θ]) (( xx ddeegg..ccmm 2.dmol -1.residue -1 ), with α-helical content calculated through deconvolution using the CDSSTR algorithm with normalised root-mean-square deviation values (nrmsd) as shown. (b, d, f) CD thermal denaturation data recorded between 5 and 95 C, in mean residue ellipticity at 222 nm ([θ] 222 ). (a) CD spectra and (b) thermal denaturation of CtIP-NTD (solid line) and CtIP-NTD-DM (dashed line). (c) CD spectra and (d) thermal denaturation of CtIP-cNTD (solid line) and CtIP-cNTD-DM (dashed line). (e) CD spectra and (f) thermal denaturation of CtIP-nNTD. (g) CtIP-NTD was analysed in buffer containing KBr. Rutherford backscattering (RBS) spectrum and (h) PIXE spectrum with raw data shown as black dots and the modelling fitting as red lines. (i) Zinc was detected at a mean atomic ratio to sulphur of 0.095, which given the presence of three methionine and three cysteine residues per chain, corresponds to a CtIP-NTD:Zn 2+ ratio of 1.8:1.
5 Supplementary Figure 3 SEC-MALS analysis of MBP-CtIP-nNTD and F20E CtIP-NTD. (a) MBP-CtIP-nNTD eluted in a single peak corresponding to a molecular weight of 187 kda; its theoretical tetramer size is 197 kda. For comparison, free MBP eluted in a single peak of 44.5 kda, corresponding to its theoretical monomer size of 44.7 kda. The light scattering (LS) as relative Raleigh ratio and the differential Reflective Index (dri) are drawn as solid and dashed lines, respectively. The value for the fitted molecular mass (M r ) of MBP-CtIP-nNTD is shown as diamond shapes across its elution peak. The predicted molecular masses of monomeric MBP-CtIP-nNTD and free MBP are shown in brackets above the respective elution peak. (b) F20E CtIP-NTD forms two distinct species of 62.3 kda and 33.2 kda, corresponding to its theoretical tetramer and dimer sizes of 63.2 kda and 31.6 kda, respectively. The light scattering (LS) as relative Raleigh ratio and the differential Reflective Index (dri) are drawn as solid and dashed lines, respectively. The values for the fitted molecular masses (M r ) of dimeric and tetrameric F20E CtIP-NTD are shown as diamond shapes across the respective elution peaks. The predicted molecular mass of monomeric F20E CtIP-nNTD is shown in brackets above the elution peaks.
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7 Supplementary Figure 4 Experimental procedure and gating scheme for the TLR system. (a) TLR cells are not fluorescent due to the fact that the TLR cassette contains an in-frame truncated version of the egfp gene and fulllength mcherry is out of frame. When TLR cells are transfected with an I-SceI nuclease encoded in a plasmid also expressing an infrared fluorescent protein (IFP) and an exogenous donor template containing the missing part of egfp that also expresses a blue fluorescent protein (BFP), the different repair outcomes will give different fluorescent cells. If the double-strand break (DSB) is repaired by gene conversion (homologous recombination), egfp is restored and the cells fluoresce in green. If the DSB is repaired by mutagenic end joining causing a +2 frameshift, mcherry gets in frame and its expression yields red fluorescence. The T2A dis-linker between egfp and mcherry allows the downstream-encoded mcherry to escape degradation of the misfolded protein encoded in the +3 reading frame of egfp. (b) Experimental outline of the TLR assay. (c-h) Gating scheme for a TLR assay. (c) Discrimination of cells over debris using a forward scatter vs. side scatter plot (areas). (d) Discrimination of cell singlets over doublets using a forward scatter (area) vs. side scatter (width) plot. (e) Selection of events for quantification. Dot plot showing the intensity of BFP (donor; x axis) vs. intensity of IFP (nuclease; y axis). Cells with negative staining for both BFP and IFP are depicted in (f) to show the absence of egfp or mcherry fluorescence in non-transfected cells, and to establish quantification gates. Cells counted positive for both BFP and IFP (that is, transfected with both the donor and the nuclease) are represented in (g), where quantification gates are the same as the ones established in (f). At least 10,000 events were analysed in the double-positive population. (h) Due to the fact that the amount of donor transfected in the cells affects the outcome of the assay, the intensity of the BFP signal in the double-positive cells (geometric mean of BFP signal in BFP+ IFP+ cells) was calculated for normalisation purposes (see Methods). (i) Mutagenic end-joining (mutej) rates in the TLR-CtIP complementation system. Only expression of the wild-type version of FLAG-CtIP reduced the rate of mutej to values similar to the control (cells transfected with control sirna). EV: empty vector. All quantifications are shown as the average of three independent experiments. Error bars are ± SEM.
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9 Supplementary Figure 5 CtIP expression levels in the different stable cell lines generated and fluorescence-activated cell sorting quantification of GFP-CtIP accumulation on damaged cells. (a) CtIP expression levels in the different U2OS-MMEJ FLAG-CtIP clones. Samples were collected 48 h after sirna transfection. CtIP antibody was a gift from R. Baer. PARP-1 antibody (Cell Signalling) was used as loading control. (b) GFP-CtIP expression levels in the U2OS stable cell lines. RPA34-20 (Merck) antibody was used as loading control. (c) A representative complete dataset for quantification of GFP-CtIP accumulation on damaged cells. Dot plots representing, for each dataset, gating and quantification (in the y axis) of the GFP-positive cells (top panels) and the γh2ax-positive cells (bottom panels) taking into account their DNA content (x axis). Quantification gates were always established using the untreated samples of each dataset. At least 10,000 events were analysed per sample.
10 Supplementary Figure 6 A representative complete dataset for the DNA-end resection assay. Dot plots representing, for each dataset, gating and quantification (in the y axis) of the RPA-positive cells (top panels) and the γh2axpositive cells (bottom panels) taking into account their DNA content (x axis). Quantification gates were always established using the untreated samples of each dataset and were defined as to avoid quantification of RPA chromatinization due to DNA replication. Note that as camptothecin (CPT) only causes DNA double-strand breaks in S-phase cells, both RPA- and γh2ax-positive cells only appear in that cell cycle stage. At least 10,000 events were analyzed per sample.
11 Supplementary Figure 7 CtIP-CTD, but not CtIP-NTD, interacts with double-stranded DNA. Electrophoretic mobility shift assay measuring the ability of CtIP NTD and CTD regions to interact with linear, double-stranded DNA. In the experiment, increasing amounts (0, 0.5, 1.0, 2.0, 5.0 and 10µM) of the CtIP protein were incubated with a 1µM sample of 5 fluorescein-labeled 200bp double-stranded DNA. After incubation, each sample was resolved by electrophoresis on agarose gel and visualised under UV light.
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Gene Forward Primer Reverse Primer GAPDH ATCATCCCTGCCTCTACTGG GTCAGGTCCACCACTGACAC SSB1 AACTTCAGTGAGCCAAACCC GTTCTCAGAGGCTGGAGAGG
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Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1
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(phosphatase tensin) domain is shown in dark gray, the FH1 domain in black, and the
Supplemental Figure 1. Predicted Domain Organization of the AFH14 Protein. (A) Schematic representation of the predicted domain organization of AFH14. The PTEN (phosphatase tensin) domain is shown in dark
SUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION doi:10.1038/nature09937 a Name Position Primersets 1a 1b 2 3 4 b2 Phenotype Genotype b Primerset 1a D T C R I E 10000 8000 6000 5000 4000 3000 2500 2000 1500 1000 800 Donor (D)
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Supplementary Figure 1 Analyses of ECTRs by C-circle and T-circle assays. (a) C-circle and (b) T-circle amplification reactions using genomic DNA from different cell lines in the presence (+) or absence