Fig. 3.

Poly-SUMO-fused Topoisomerase II variants have distinct biological properties in vivo. a Ectopic expression of Top2p–CSM in diploid cells. Western blotting of Top2p–CSM–HA construct integrated into W303 diploid cells (See Table 2, AS611–AS618). The control lane shows wild-type Top2p–HA in haploid cells (1033-W303-1A; not ectopic). b Fused SUMO molecules are counted toward total Top2p sumoylation in vitro. Sumoylation of chromatin-bound Top2p in vitro is described in detail in Takahashi et al. (2006). Briefly, the purified recombinant components of the conjugation reaction (Takahashi et al. 2003) were used in the reaction mixture containing chromatin-bound Top2p–HA (1033-W303-1A strain), Top2p–1Smt3–HA (AS611 segregant), Top2p–2Smt3–HA (AS612 segregant), and Top2p–3Smt3–HA (AS613 segregant), as substrates (right panel). The mixture kept on ice (Incub. −) was used as a negative control (left panel). Reaction products were analyzed by Western blotting using anti-HA antibody. The arrow points to the upper limit of sumoylation (five to six Smt3p), which is applicable to all tested constructs. c Top2p with long SUMO CSM chains inhibits cell growth. Meiotic progeny tetrads from diploid strains shown in a. Two dissected tetrads (30°C, 3 days) are shown for diploids expressing Top2p–SNM–HA (AS619), control, Top2p–1Smt3 (AS611), Top2p–2Smt3 (AS612), and Top2p–7Smt3 (AS617). The inhibitory effect of Top2p–7Smt3 results in very small colonies (last panel). A similar inhibitory effect is detected starting from 4× SUMO fused (not shown). d Four-SUMO threshold for stable maintenance of Top2p–CSM constructs. Western blotting showing extracts from haploid strains expressing Top2p–CSM constructs as a sole source of Topoisomerase II: Top2–Smt3 (a AS611 segregant), Top2p–2Smt3 (AS612 segregant), Top2p–3Smt3 (AS613 segregant), Top2p–4Smt3 (AS614 segregant), Top2p–5Smt3 (AS615 segregant), Top2p–6Smt3 (AS616 segregant), and Top2p–8Smt3 (AS618 segregant). The complete loss of all tandem SMT3 copies but one is evident for 5× (AS615 segregant) and above constructs, while the 4× (AS614 segregant) construct has only traces of full-length fusion (dashed arrow). The 8× construct in the haploid strain (AS618 segregant) results in the rapid loss of the whole integrated plasmid (including the HA epitope tag). e The necessity to reduce SUMO numbers by recombination results in reduced growth rates for 4–8× Top2p–CSM constructs. Fresh segregants containing Top2p–CSM with various numbers tandem SUMO were spotted onto YPD plates and incubated for 2 days. The growth-inhibitory effects are evident for 4 and 8× constructs, which eliminate excessive CSM SUMO by different recombination pathways. 5 and 6× constructs, which rapidly pop-out excessive CSM SUMO, show little growth inhibition in this analysis