Journal: eLife

Article Title: DNA-Stimulated Liquid-Liquid phase separation by eukaryotic topoisomerase ii modulates catalytic function

doi: 10.7554/elife.81786

Figure Lengend Snippet: Figure 4. Ionic, protein-DNA interactions drive LLPS by ScTop2. (A) Representative images of 1µM Cy3-ScTop2ΔCTD in 150mM KOAc and 1μM ScTop2CTD in 50mM KOAc mixed with and without 50nM Cy5-200 bp DNA. (B) Representative images of 500nM Cy3-ScTop2 mixed with varying concentrations of pSG483 in the presence of different buffer conditions. Control reaction has 150mM KAcetate. Salt concentrations represent the final values in solution. 1,6-hexanediol (1,6-HD) weight by volume percent was added to 150mM KAcetate solution. (C) Representative images of puncta/fibrils comparing phosphatase-treated and untreated ScTop2. (D) List of phosphorylated amino acids as identified by mass spectrometry analysis. (E) FRAP analysis of phosphatase-treated protein. (F) Quantification of mobile phase for Figures 2B and 3E Error bar is standard deviation for each condition. Two-Way ANOVA with bonferroni post hoc analysis was done on ten puncta per condition. ns is non-significant, **** means p<0.0001. Scale bars in (A), (B), and (C) are 5µm.

Article Snippet: The ScTop2CTD (1178–1428) expression plasmid was cloned by PCR amplification from full- length ScTop2 12URA- B vector followed by insertion into a modified 1- B (Addgene #29653) E. coli expression vector.

Techniques: Control, Mass Spectrometry, Standard Deviation

Journal: eLife

Article Title: DNA-Stimulated Liquid-Liquid phase separation by eukaryotic topoisomerase ii modulates catalytic function

doi: 10.7554/elife.81786

Figure Lengend Snippet: Figure 5. The ScTop2 CTD modulates protein catenation versus knotting. (A) Activity assay at different ScTop2 concentrations with 25nM pSG483 and either 150mM or 400mM KAcetate. LLPS does not occur at the higher salt concentration. Confocal images were taken before the addition of ATP and with Cy3-labeled protein. ScTop2 concentration in the ‘no ATP’ reaction is 1μM. No ATP and no topoisomerase reactions are shown as negative controls. (B) Modified activity assay with 1μM ScTop2 and 25nM pSG483. Workflow is shown at right. (C) Activity assay performed with ScTop2ΔCTD and

Article Snippet: The ScTop2CTD (1178–1428) expression plasmid was cloned by PCR amplification from full- length ScTop2 12URA- B vector followed by insertion into a modified 1- B (Addgene #29653) E. coli expression vector.

Techniques: Activity Assay, Concentration Assay, Labeling, Modification