Figure 6.

Mutations in the Hinge Domain of Smc1/Smc3 Destroy Cohesin's Ability to Coentrap Sister Minichromosomes

Exponential-phase cells of strains K16338 (MATa, td-SMC1, YIplac211::URA3), K16335 (td-SMC1, SMC1-Myc9::URA3), and K16339 (MATa, td-SMC1, smc1(M665R)-Myc9::URA3) or K16341 (MATa, td-SMC3, YIplac211::URA3), K16342 (MATa, td-SMC3, SMC3-HA3::URA3), and K16343 (MATa, td-SMC3, smc3(M577K)-HA3::URA3) harboring a 7.5 kb minichromosome were arrested with α factor at 25°C. Endogenous Smc1 or Smc3 was degraded by shifting cultures to degron-mediated proteolysis conditions (YEP gal, 5 μg/ml doxycycline [DOX], 37°C) for 1 hr. Subsequently, the cells were released from G1 arrest to YEP gal medium containing 5 μg/ml doxycycline and 10 μg/ml nocodazole. After 100 min at 37°C, the cells were lysed and extracts were fractionated by sucrose gradient centrifugation followed by gel electrophoresis. Minichromosome DNA was detected by Southern blotting.

(A) FACS analysis shows that all strains completed DNA replication in the above conditions.

(B) Western blot (right) showing that endogenous HA-tagged td-Smc1 proteins are completely degraded and hence that ectopically expressed Myc-tagged Smc1 is the only version of Smc1 in the minichromosome cohesion assay (left). In the td-Smc3 strains, endogenous HA-tagged td-Smc3 proteins are also completely degraded, and ectopically expressed HA-tagged Smc3 proteins are the only version of Smc3.

(C) Minichromosome DNA was detected by Southern blotting. Monomers and dimers are marked inside the red and green boxes, respectively.