Journal: The Journal of Biological Chemistry

Article Title: Casein kinase II–dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast

doi: 10.1074/jbc.RA118.004955

Figure Lengend Snippet: Top2–2A (S1363A,S1364A) mutant protein maintains decatenation activity but has reduced ATPase activity. A , SDS-PAGE patterns of WT Top2 and alanine substitution mutant Top2-2A (S1363A and S1364A). FLAG-tagged Top2-WT and -2A proteins were overproduced under the inducible nmt promoter (plasmid Rep41) in WT S. pombe cells and immunoprecipitated using anti-FLAG antibody. A strain containing only the vector was used as a control. Immunoprecipitated FLAG-tagged Top2 proteins are indicated by arrows . The position of the protein marker bands ( M ) is indicated. B , Top2 decatenation assay. kDNA (153 ng) was incubated with immunoprecipitated Top2 fractions in ATP-containing reaction buffer (see “Experimental procedures”) for 1–30 min at 37 °C. An immunoprecipitated fraction from cell extracts containing only the empty vector was used for a mock reaction ( vector IP ). The reaction was terminated using stop buffer and loaded onto a 1% agarose gel followed by ethidium bromide staining. Only the catenated kDNA ( kDNA ) and decatenated kDNA ( decat. kDNA ) were loaded as controls, along with λDNA digested by EcoT14I (λ -EcoT14I ). Positions of catenated and decatenated kDNA are indicated by an arrow and a vertical line , respectively. The ratio of decatenated DNA to total DNA (catenated + decatenated) was quantified. Phosphorylation of Ser 1363 and Ser 1364 does not affect Top2 decatenation activity. C , Top2 ATPase assay. Immunoprecipitated Top2-WT or -2A mutant proteins were incubated with ATP and kDNA in the presence or absence of the 5 μ m anti-cancer topo II inhibitor ICRF-193 for 30 min at 30 °C. Free phosphate produced by ATP hydrolysis was measured by malachite green colorimetric reagent (see “Experimental procedures”). Error bars represent the standard deviation for each experiment performed in triplicate. p values for comparison among four conditions were calculated using one-way analysis of variance with Holm multi-comparison correction. *, p < 0.05.

Article Snippet: Top2 decatenation assays were performed using the Topoisomerase II Assay Kit (TopoGEN, Inc., TG1001-1).

Techniques: Mutagenesis, Activity Assay, SDS Page, Plasmid Preparation, Immunoprecipitation, Control, Marker, Incubation, Agarose Gel Electrophoresis, Staining, Phospho-proteomics, ATPase Assay, Produced, Standard Deviation, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Casein kinase II–dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast

doi: 10.1074/jbc.RA118.004955

Figure Lengend Snippet: Defective chromosome segregation induced by an anti-cancer catalytic topo II inhibitor, ICRF-193, is exacerbated in cka1-372 and top2-2A (S1363A,S1364A) mutant cells. A , CKII ts mutant cka1-372 cells showed more severe defects in mitotic chromosome segregation than WT cells in the presence of ICRF-193. Left panel , DAPI-stained micrographs of WT and cka1-372 mutant cells were obtained at the restrictive temperature (36 °C) for 3 h in the presence of DMSO or ICRF-193 (5 and 10 μ m ). A displaced nuclear phenotype was frequently observed in ICRF-193–treated cka1 mutant cells ( arrows ). Right panel , frequencies of defective phenotypes categorized as lagging-like ( blue ), streaked chromosomes ( red ), and displaced nucleus ( green ). More than 200 anaphase cells were counted for each sample. Error bars represent the standard deviation for each defective phenotypes. p values for comparison between the drug-treated WT and cka1 mutant were calculated using a Student's t test. *, p < 0.05; **, p < 0.01 ( black , total frequency of abnormal phenotypes; color , each phenotype). B , defects in chromosome segregation increased significantly in unphosphorylatable top2-2A mutants compared with WT and phosphomimetic top2–2E mutant cells in the presence of ICRF-193. Cells were asynchronously cultured at 26 °C for 2 h in the presence of DMSO or 5 μ m ICRF-193. Left panel , representative micrographs of DMSO- and 5 μ m ICRF-193–treated top2-2A cells. Chromatin DNA was stained with DAPI. Abnormally streaked chromosomes in anaphase are indicated ( arrows ). Scale bar = 10 μm. Right panel , frequencies of anaphase cells with abnormally streaked chromosomes. More than 200 anaphase cells were counted for each sample. Error bars represent the standard deviation for each experiment performed in biological triplicates. Significant differences among the three strains were examined using one-way analysis of variance with Bonferroni multi-comparison correction. *, p < 0.05; **, p < 0.01; n.s. , not significant.

Article Snippet: Top2 decatenation assays were performed using the Topoisomerase II Assay Kit (TopoGEN, Inc., TG1001-1).

Techniques: Mutagenesis, Staining, Standard Deviation, Comparison, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Casein kinase II–dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast

doi: 10.1074/jbc.RA118.004955

Figure Lengend Snippet: Cartoon of the possible action of phosphorylated topo II residues Ser 1363 and Ser 1364 on the ATP-binding and hydrolysis domain targeted by ICRF-193. An ATP-binding hydrolytic domain ( green ), a DNA-binding cleavage domain ( light blue ), and a C-terminal region ( blue ) are illustrated with gate ( G ) and transport ( T ) segments of dsDNAs. The T-shaped line represents the inhibition of the ATP-binding hydrolytic domain by ICRF-193 (a structural formula is shown). The dashed arrow represents the possible action of the topo II residues Ser 1363 and Ser 1364 phosphorylated by CKII. See text for more details.

Article Snippet: Top2 decatenation assays were performed using the Topoisomerase II Assay Kit (TopoGEN, Inc., TG1001-1).

Techniques: Binding Assay, Inhibition