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. 2015 Jan 1;26(1):117–133. doi: 10.1091/mbc.E14-08-1268

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© 2015 Guacci et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 6: — smc3-D1189H robustly suppresses the cohesion defect of the smc3-K112R, K113R (RR) mutation. (A) Plasmid shuffle to assess viability of the chimeric smc3-RR-D1189H allele. Haploid shuffle strain VG3464-16C bearing plasmid pEU42 (SMC3 CEN URA3) and a second SMC3 “test allele,” SMC3, smc3-D1189H, smc3-RR, or chimeric smc3RR-D1189H, was grown and plated as described in Figure 2A onto URA–dropout or FOA-containing media. Plates were incubated 3 d at 23°C. (B) Assessment of drug sensitivity. Haploid SMC3 (MB45-1A), smc3-D1189H (MB46-1A), or chimeric smc3-RR-D1189H (MB47-1A) strains grown and plated as described in Figure 2A onto YPD, BEN, and CPT and incubated at 30°C for 2 d. (C). Cohesion loss of in smc3-RR cells. Haploids bearing SMC3-AID and a second SMC3 allele, either WT or smc3-RR, were depleted for SMC3-AID from G1 through mid–M phase arrest. Left, cohesion loss at CEN-proximal TRP1 locus assessed in haploid SMC3 SMC3-AID (MB84-1A) and smc3-RR SMC3-AID (MB83-1A) strains. Right, cohesion loss at CEN-distal LYS4 locus assessed in haploid SMC3 SMC3-AID (MB81-1A) and smc3-RR SMC3-AID (MB79-1A) strains. The percentage of cells with two GFP spots (sister separation) is plotted. (D) Cohesion loss of smc3-RR-D1189H in mid–M phase–arrested cells. Haploid SMC3, smc3-D1189H and chimeric smc3-RR-D1189H were arrested in mid–M phase as described in Figure 1B. Left, cohesion loss at CEN-proximal TRP1 locus assessed in haploid wild-type (SMC3; MB65-1A), smc3-D1189H (MB66-1A), or chimeric smc3-RR-D1189H (MB67-1A) strain. Right, cohesion loss at CEN-distal LYS4 locus assessed in haploid strains from B. The percentage of cells with two GFP spots (sister separation) is plotted. (E, F) Assessing smc3-D1189H ability to suppress Eco1p depletion. (E) Viability after Eco1p depletion. Haploid WT (VG3620-4C), ECO1-AID (VG3662-1D), and smc3-D1189H ECO1-AID (VG3663-2E) were grown and plated as described in Figure 2A onto YPD alone or containing auxin (750 μM) and then incubated at 23°C for 2 d. (F) Cohesion loss after auxin-mediated Eco1p depletion from G1 cells through mid–M phase arrest. Left, cohesion loss at CEN-proximal TRP1 locus assayed in haploid strains WT (VG3460-2A), ECO1-AID2 (VG3659-1A) and smc3-D1189H ECO1-AID2 (VG3663-2E) strains. Right, cohesion loss at CEN-distal LYS4 locus assessed in haploid WT (VG3620-4C), ECO1-AID2 (VG3646-1A) and smc3-D1189H ECO1-AID2 (VG3650-1E) strains. The percentage of cells with two GFP signals (sister separation) is plotted. (C, D, F) Data were derived from two independent experiments; 100–300 cells were scored for each data point in each experiment.