Journal: Cancer cell

Article Title: Phosphorylation of ETS1 by Src Family Kinases Prevents its Recognition by the COP1 Tumor Suppressor

doi: 10.1016/j.ccr.2014.06.026

Figure Lengend Snippet: (A) Schematics of ETS1 and ETS2.

Article Snippet: Anti-COP1 antibody (A300-894A), anti-ETS1 antibody (A303-501A), anti-ZEB2 antibody (A302-474A), anti-V5 antibody (A190-120A), HRP conjugated anti rabbit secondary antibody (ReliaBLOT, WB120) were purchased from Bethyl.

Techniques:

Journal: Cancer cell

Article Title: Phosphorylation of ETS1 by Src Family Kinases Prevents its Recognition by the COP1 Tumor Suppressor

doi: 10.1016/j.ccr.2014.06.026

Figure Lengend Snippet: (A and B) Immunoblot analysis of anti-ETS1 immunoprecipitates or whole cell extracts of MDA-MB-231 cells expressing a DOX-inducible COP1 shRNA treated with increased amounts of dasatinib for 8 hours with or without pretreatment of MG132 (10 μM) for 2 hours (A) or Dox (1μg/mL) for 48 hours (B).

Article Snippet: Anti-COP1 antibody (A300-894A), anti-ETS1 antibody (A303-501A), anti-ZEB2 antibody (A302-474A), anti-V5 antibody (A190-120A), HRP conjugated anti rabbit secondary antibody (ReliaBLOT, WB120) were purchased from Bethyl.

Techniques: Western Blot, Expressing, shRNA

Journal: Cancer cell

Article Title: Phosphorylation of ETS1 by Src Family Kinases Prevents its Recognition by the COP1 Tumor Suppressor

doi: 10.1016/j.ccr.2014.06.026

Figure Lengend Snippet: (A and B) Immunoblot analysis (A) and soft agar assay (B) of MDA-MB-231 cells stably expressing shRNAs against GFP or ETS1 (shRNA 2 or 3) and, where indicated, an mRNA encoding venus fluorescent protein (Venus) or an shRNA-resistant (for shRNA 3) mRNA encoding ETS1 (ETS1res). Representative images in (B) are from three biological replicates.

Article Snippet: Anti-COP1 antibody (A300-894A), anti-ETS1 antibody (A303-501A), anti-ZEB2 antibody (A302-474A), anti-V5 antibody (A190-120A), HRP conjugated anti rabbit secondary antibody (ReliaBLOT, WB120) were purchased from Bethyl.

Techniques: Western Blot, Soft Agar Assay, Stable Transfection, Expressing, shRNA

Journal: Nucleic Acids Research

Article Title: Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

doi: 10.1093/nar/gkw228

Figure Lengend Snippet: Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts. ( A ) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. ( B ) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. Phosphotyrosyl bond hydrolysis catalyzed by mTdp2 cat releases p -nitrophenol, which is detected by measuring absorbance at 415 nm. ( C ) mTdp2 cat reaction rates on p –nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s −1 produced by mTdp2 cat . P -values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. ( D ) Structure of mTdp2 cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. PDB entry 5HT2 is displayed, also see Table . ( E ) Structure of mTdp2 cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. PDB entry 5INK is displayed, also see Table . ( F ) Structure of mTdp2 cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. PDB entry 5INM is displayed, also see Table .

Article Snippet: Expression of human Tdp2 (hTdp2 FL ) was validated by Western analysis (12203-1-AP; Proteintech).

Techniques: Derivative Assay, Synthesized, Modification, Produced, Two Tailed Test, Binding Assay

Journal: Nucleic Acids Research

Article Title: Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

doi: 10.1093/nar/gkw228

Figure Lengend Snippet: Data collection and refinement statistics

Article Snippet: Expression of human Tdp2 (hTdp2 FL ) was validated by Western analysis (12203-1-AP; Proteintech).

Techniques:

Journal: Nucleic Acids Research

Article Title: Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

doi: 10.1093/nar/gkw228

Figure Lengend Snippet: Structure-function analysis of the Tdp2 reaction mechanism. ( A ) Proposed mechanism for hydrolysis of phosphotyrosine bond by Tdp2. Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3. Residue numbers shown are for the mTdp2 homolog. ( B ) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom. Reaction proceeds from right to left. ( C ) Models for the mTdp2 cat -DNA complex during the QM/MM reaction path simulation showing the substrate (left, tan), transition state intermediate (center, cyan) and product (right, pink) states. Residue numbers shown are for the mTdp2 homolog. ( D ) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). ( E ) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2 cat fusion proteins on the three substrates. Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm. Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2 cat ; error bars, s.d. n = 3. Mutants of hTdp2 (black) and the equivalent residue in mTdp2 (tan) are indicated.

Article Snippet: Expression of human Tdp2 (hTdp2 FL ) was validated by Western analysis (12203-1-AP; Proteintech).

Techniques: Binding Assay, Residue, Isolation, Activity Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

doi: 10.1093/nar/gkw228

Figure Lengend Snippet: Tdp2 SNPs impair function. ( A ) Active site residues mutated by TDP2 SNPs. D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2 cat structure (4GZ1). ( B ) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2 cat proteins used for assays in panels C and D. ( C ) Activity of WT and mutant MBP-hTdp2 cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. Samples were withdrawn from reactions, neutralized with TBE-urea loading dye at the indicated timepoints, and electrophoresed on a 20% TBE-urea PAGE. ( D ) Relative activity of WT and indicated mutant human MBP-hTdp2 cat fusion proteins on three model Tdp2 substrates. Quantification of percent MBP-hTdp2 cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure . Error bars, s.d. n = 3.

Article Snippet: Expression of human Tdp2 (hTdp2 FL ) was validated by Western analysis (12203-1-AP; Proteintech).

Techniques: Staining, SDS Page, Purification, Mutagenesis, Activity Assay

Journal: Nucleic Acids Research

Article Title: Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

doi: 10.1093/nar/gkw228

Figure Lengend Snippet: Metal cofactor interactions with Tdp2. ( A ) Intrinsic tryptophan fluorescence of mTdp2 cat was used to monitor a conformational response to divalent metal ion binding. Either Mg 2+ or Ca 2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. Both Mg 2+ and Ca 2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2 cat in the presence and absence of DNA, while D358N active site mutant of mTdp2 cat is unresponsive to Mg 2+ . ( B ) mTdp2 cat activity assayed on a T5PNP substrate as a function of Mg 2+ and Ca 2+ concentration. PNP release (monitored by absorbance at 415 nm) as a function of Mg 2+ concentration and in the absence or presence of 1 or 10 mM Ca 2+ is shown; error bars, s.d. n = 4. ( C ) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2 cat –DNA–Mn 2+ complex (PDB entry 5INP) show a single Mn 2+ (cyan) is bound with expected octahedral coordination geometry. A 53σ peak in the anomalous difference Fourier map (data collected at λ = 1.5418 Å) supports Mn 2+ as the identity of this atom. ( D ) Comparison of Ca 2+ (green Ca 2+ ion, orange DNA) (PDB entry 5INQ), and Mg 2+ (magenta Mg 2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2 cat –DNA structures shows that Ca 2+ distorts the 5′-phosphate binding mode.

Article Snippet: Expression of human Tdp2 (hTdp2 FL ) was validated by Western analysis (12203-1-AP; Proteintech).

Techniques: Fluorescence, Binding Assay, Mutagenesis, Activity Assay, Concentration Assay, Comparison

Journal: Nucleic Acids Research

Article Title: Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

doi: 10.1093/nar/gkw228

Figure Lengend Snippet: Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. ( A ) Cy5 labeled substrates with 5′-phosphate termini ( Lanes 1–4) or 5′-tyrosylated termini ( Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2 FL as indicated (+) for 5 min at 37°C. Concatemer ligation products were detected by 5% native PAGE. ( B ) Workflow diagram of cellular end joining assays. DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells. After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. ( C ) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2 −/− cells and Tdp2 −/− cells complemented with wildtype or the noted hTDP2 FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. Error bars, s.d, n = 3. ( D ) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate. Error bars, s.d, n = 3. ( E ) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3.

Article Snippet: Expression of human Tdp2 (hTdp2 FL ) was validated by Western analysis (12203-1-AP; Proteintech).

Techniques: Labeling, Incubation, Ligation, Clear Native PAGE, Cell Culture, Sequencing, Clonogenic Cell Survival Assay, Knock-Out

Journal: Nucleic Acids Research

Article Title: Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

doi: 10.1093/nar/gkw228

Figure Lengend Snippet: Conformational plasticity in the Tdp2 active site. ( A ) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2 cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. Wall-eyed stereo view is displayed. ( B ) The closed β2Hβ conformation in the mTdp2 cat –DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. Wall-eyed stereo view is displayed. ( C ) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2 cat (PDB entry 5INM, see Table ) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). ( D ) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. mTdp2 cat WT (lanes 1–13) or mTdp2 cat D358N (lanes 14–26) were incubated in the presence or absence of Mg 2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl −1 of trypsin. Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. ( E ) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop. Experiments performed as in panel D for mTdp2 cat WT (lanes 27–39) or mTdp2 cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin.

Article Snippet: Expression of human Tdp2 (hTdp2 FL ) was validated by Western analysis (12203-1-AP; Proteintech).

Techniques: Solvent, Binding Assay, Sequencing, Incubation, SDS Page, Staining