Fig. S3.

Effect of the eco1^W216G mutation on cohesion. (A) Analysis of a CEN/ARS minichromosome. A GFP-tagged, CEN/ARS minichromosome (pJSC2) was constructed in yeast by joining plasmids pRS416 and pSR10 via in vivo recombination, according to the method of ref. 52. pJSC2 is comparable in size to DNA circles bearing either HMR or URA3. Stains JSC209 (wt), JSC81 (eco1^W216G), and JSC208 (smc3-42) containing pJSC2 were grown overnight in SC-trp plus dextrose at 25 °C. An equal volume of YPAD was added when the cultures reached midlog, and 20 min later, nocodazole was added. After 3 h, half of the culture was shifted to 37 °C. Cohesion was evaluated after 2 additional hours. The data show that cohesion of a circular, CEN/ARS minichromosome was not diminished by eco1^W216G. (B) Analysis of HMR. DNA circles bearing HMR were examined in nocodazole-arrested strains RDY215 (wt) and JSC52 (eco1^W216G) at 25 °C per the cohesion assay. To examine the unrecombined chromosome, cells were grown to midlog in dextrose at 25 °C and arrested with nocodazole. The data show that cohesion of HMR DNA circles was abolished in the eco1^W216G mutant, whereas cohesion of the unrecombined chromosomal arm was not.