Figure 3.

Increased Levels of Top2 Promote SCI Formation

(A) A strain carrying a chromosomal insertion of GAL-TOP2 in the ura3 locus and a wild-type strain were grown and plated on media containing glucose (where GAL-TOP2 is not expressed) or galactose (where GAL-TOP2 is expressed) at 30°C. Images were taken after 3 days. Expression of TOP2 from the GAL1-10 promoter impaired cellular growth.

(B) Wild-type and GAL-TOP2 strains bearing the 10 kb circular minichromosome were synchronized in G₁ in media containing raffinose and released into the cell cycle in the presence of nocodazole at 30°C. Following metaphase arrest, cells were transferred to new media containing galactose and nocodazole to activate expression of TOP2 from the GAL1-10 promoter. Samples were taken for analysis just before and 120 min after transfer to galactose. Cell extracts were separated by differential sedimentation, heat denatured in 1% SDS, and analyzed by Southern blotting to reveal plasmid species. Plasmid species included monomeric (OCm, Lm, and CCCm) and dimeric forms, including different SCI species (Catc-type catenanes and Catb-type catenanes). Running position for SCIs in the blots are indicated (dashed boxes). SCIs (including c- and b-type catenanes) were quantified using ImageJ software (right-hand graphs; showing the mean ± SD from three independent experiments). SCIs increased upon activation of GAL-TOP2.

(C) GAL-TOP2 and control strain bearing a circular minichromosome (pRS316) were blocked in G₁ in media containing raffinose and released into the cell cycle in the presence of galactose and nocodazole at 30°C. DNA was purified from cells in the G₁ arrest (G₁− Raffinose) and at the metaphase block (M−Galactose) and analyzed by two-dimensional gel chloroquine analysis to assess the supercoiling status of monomer plasmids in the cells. A small distribution change toward a less negatively supercoiled population was observed upon TOP2 expression (bottom panels). A cartoon representation of how the plasmid distribution relates to supercoiling status is shown.

(D) GAL-TOP2 bearing a circular minichromosome (pRS316) was blocked in G₁ in media containing raffinose and released to nocodazole in raffinose media at 30°C. Upon metaphase arrest, half of the culture was maintained in raffinose while galactose was added to the other half to induce TOP2 expression. Samples were taken 120 min after the galactose addition. DNA was purified and analyzed as in (C). A small distribution change toward a less negatively supercoiled population was observed upon TOP2 expression.