Fig 1: Human TDP1 is SUMOylated in vitro and in mammalian cells.(a) Yeast Y190 cells containing the indicated constructs were examined for the activation of His3 and β-Gal reporter genes. (b) Recombinant p53 (430 nM) was incubated with 50 nM SAE1/SAE2, 500 nM UBC9, 5 mM ATP and 30 μM SUMO1 'WT' or mutant SUMO1 that cannot be covalently conjugated 'MT'. Reactions were fractionated by SDS–PAGE and analysed by anti-p53 immunoblotting (Activemotif). (c) Human full-length TDP1 (500 nM) was subjected to in vitro SUMOylation reactions and analysed by anti-TDP1 (ab4166; Abcam) or anti-SUMO1 (Santa Cruz, SC-5308) immunoblotting. (d) SUMOylation reactions were conducted in the presence or absence of 5 mM ATP. (e) SUMOylation reactions conducted in the presence of SUMO1, SUMO2 or SUMO3 were divided into three fractions and analysed by immunoblotting. (f) Total cell extract from HEK293 (∼4×106 cells) transfected with Myc–TDP1 and/or GFP–SUMO1 was subjected to immunoprecipitation using anti-Myc monoclonal antibodies (9B11; Cell Signaling). Immunoprecipitates were analysed by immunoblotting using anti-Myc (9B11) or anti-SUMO1 (SC-5308) antibodies. Input is ∼5% of total cell extract. (g) HEK293 cells transfected with Myc–TDP1, HA-tagged His–SUMO1, His–SUMO2 or His–SUMO3 were analysed by anti-TDP1 (ab4166) or anti-His (H1029) immunoblotting. (h) Lysates from control HeLa cells 'C' or cells stably expressing His–SUMO1 'S1' were mixed with Ni2+ charged agarose beads and histidine-tagged SUMO conjugates 'bound' were analysed by anti-TDP1 immunoblotting. Input is ∼3% of total cell extract. (i) Total cell extract 'In' (∼20 μg) or Ni2+ beads enriched with histidine-tagged SUMO conjugates 'bound' were mixed with 32P-radiolabelled 18-mer duplex (50 nM) harbouring a 3′-phosphotyrosine terminus 'PY', inset. Repair products were analysed by denaturing PAGE and phosphorimaging. Positions of the 32P-radiolabelled substrate 'PY' and product 'P' are indicated. (j) Diagram depicting purification of SUMOylated TDP1 from mammalian cells. (k) Serial dilutions of purified Myc–TDP1 or Myc–TDP1–SUMO1–His–GFP were analysed by SDS–PAGE and anti-Myc immunoblotting. (l) Purified Myc-TDP1–SUMO1–His–GFP was mock-treated '−' or incubated with 250 ng of human sentrin/SUMO-specific protease-1 (SENP1) '+' and reaction products analysed by anti-Myc immunoblotting. Molecular weight size markers (kDa) are depicted. IB, immunoblot; IgG, immunoglobulin G; MT, mutant; WT, wild type.
Fig 2: TDP1 is SUMOylated at Lysine 111.(a,b) BLAST analysis identifies TDP1 orthologues in plants, fungi and animals with an extended amino terminus in vertebrates 'dotted boxes'. Sequence analysis of this domain identifies two putative SUMOylation sites. Alignment of sequences from human (Homo sapiens; NP_001008744), cattle (Bos taurus; NP_001180084; XP_874680), monkey (Pongo abelii; XP_002825063), chicken (Gallus gallus; XP_421313), frog (Xenopus tropicalis; NP_001039242), fish (Danio rerio; XP_700174) using UniProt software identifies K111 (highlighted in grey) as a conserved residue. (c) Purified human TDP1 encompassing the first 150 amino acids TDP11−150 'WT' and equivalent versions, in which lysine 111, 139 or both were mutated to arginine 'K111R, K139R and K111R; K139R, respectively' were subjected to SUMOylation reactions containing 50 nM SAE1/SAE2, 500 nM UBC9 and 5 mM ATP in the presence of 30 μM WT SUMO1 (lanes 1–4) or mutant SUMO1 (lanes 5 and 6). Reaction products were fractionated by SDS–PAGE and analysed by immunoblotting with anti-TDP1 (ab4166; Abcam) or anti-SUMO1 (Activemotif) antibodies. (d) Purified full-length human TDP1 'WT' or TDP1K111R 'K111R' was subjected to SUMOylation reactions and analysed by immunoblotting. (e) HEK293 cells were transfected with '+' or without '−' GFP–SUMO1 and with wild-type Myc–TDP1 or a version harbouring substitution of K111 'N1', K139 'N2', K111 and K139 'N1/2', K231, K417 and K527 'C' or K111, K139, K231, K417 and K527 'NC' to arginine. Total cell extract (∼4×106 cells) was subjected to immunoprecipitation using anti-Myc monoclonal antibodies (9B11; Cell Signaling) and immunoprecipitates were fractionated by SDS–PAGE and analysed by immunoblotting. Molecular weight size markers (kDa) are depicted. MT, mutant; WT, wild type.
Fig 3: TDP1 mutant proteins. Recombinant mutant TDP1 proteins (6.25 pM) as described in the text were incubated with a final concentration of 10 nM oligonucleotide substrate over a 3-h time course. Quench reagent was then added and fluorescence signal measured. Graph represents the mean of three independent experiments. Error bars represent the standard deviation.
Fig 4: Enzyme kinetics evaluation. (A) Enzyme titration and time course of TDP1 in the Gyrasol assay. Final substrate concentration was 10 nM. (B) Kinetics of product formation for two TDP1 concentrations. Final substrate concentration was 10 nM. Graphs represent the mean of three independent experiments. Error bars represent the standard deviation.
Fig 5: The TDP1 catalytic mechanism is conserved from yeast to humanA. Overlay of yeast and human crystal protein structure showing the catalytic HxK-motif; labeled in green are human TDP1 residue numbering (PDB # 1NOP [2]) and in cyan the yeast TDP1 residue numbering (PDB# 1Q32 [4]). His263 in hTDP1 and His182 of yTDP1 represent the nucleophilic Histidine (Hisnuc), while His493 of hTDP1 and His432 in yTDP1 represent the general acid/base Histidine (Hisgab). B. TDP1 two-step catalytic mechanism. Step 1 (Formation): TDP1 resolves the DNA-adduct via nucleophilic attack of the Hisnuc that releases the adduct and generates a TDP1-DNA covalent complex via the 3’phospho-amide bond. Step 2 (Dissociation): Hisgab activates a water molecule that hydrolyze the TDP1-DNA linkage, dissociating TDP1 from the DNA, leaving behind a single-strand nick, which requires further processing by polynucleotide kinase-phosphatase prior to DNA ligation.
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