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. Author manuscript; available in PMC: 2016 Aug 8.
Published in final edited form as: Nat Struct Mol Biol. 2016 Feb 8;23(3):209–216. doi: 10.1038/nsmb.3173

Figure 4. Increasing sumoylation by Mcm6-SuOn slows growth and this defect is suppressed by a ubc9 mutant.

Figure 4

a. Mcm6-SuOn exhibits elevated sumoylation in G1 and S phases. Immunoblot at top shows Mcm6-ctrl and Mcm6-SuOn proteins (tagged with V5) from whole cell lysates. Equal loading is indicated by Ponceau stain, and lack of checkpoint activation is shown by the absence of Rad53 phosphorylation. MMS-treated sample without tagging shows no cross-reaction bands on α-V5 blot (top) and robust Rad53 phosphorylation (bottom).

b. Sumoylation of Mcm6-SuOn and Mcm6-ctrl occurs in the chromatin-bound fraction in G1 cells.

c. Sumoylation of Mcm6-SuOn in G1 requires Cdc6. Cell growth is described in Fig. 1d and Mcm6 sumoylation is detected as in (a). Cdc6 depletion is detected by immunoblotting. * indicates a cross-reaction band.

d. Mcm6-SuOn, but not Mcm6-ctrl, results in slow growth. Representative tetrads from Mcm6-SuOn/+ or Mcm6-ctrl/+ diploid strains are shown. Spores clones were grown at 30°C for 3–4 days and genotypes are indicated.

e. Slow growth of Mcm6-SuOn cells is suppressed by a SUMO E2 Ubc9 mutation (ubc9-10). Representative tetrads from dissection of Mcm6-SuOn/+ ubc9-10/+ diploid strains are shown as in (d).