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. Author manuscript; available in PMC: 2023 Mar 27.
Published in final edited form as: Cell Rep. 2021 Nov 23;37(8):110034. doi: 10.1016/j.celrep.2021.110034

Figure 2. Loss of Ulp1 tethering to the nuclear periphery suppresses tdp1Δ wss1Δ via degradation of the Siz2 SUMO ligase.

Figure 2.

(A) The role of E3 SUMO ligases in tdp1Δ wss1Δ suppression. Tetrad analysis of [TDP1/tdp1Δ; WSS1/wss1Δ; SIZ1/siz1Δ; SIZ2/siz2Δ] and [TDP1/tdp1Δ; WSS1/wss1Δ; MMS21/mms21-11] diploids. Representative spores (except mms21-11) were grown on the same plate.

(B) Siz1, Siz2, and Mms21 protein levels in ulp1-ΔN. Cells expressing E3 SUMO ligases genomically tagged with 13Myc at their C terminus were subjected to immunoblotting (IB). For quantification, anti-Myc signal was normalized to Pgk1 and then to WT.

(C) Decreased Siz2 protein levels in ulp1-ΔN are essential to rescue tdp1Δ wss1Δ. Strains were grown on SC-leu to select for plasmids expressing Siz2-13Myc (+) or p415 empty vector (−) and imaged after 72 h. See also Figure S2B for Siz2 protein levels.

(D) DNA damage checkpoint activation measured by Rad53 phosphorylation (gel shift on anti-Rad53 immunoblot).Where indicated, 1 mM auxin was added for 6 h. The anti-Pgk1 control was run in parallel on a different gel.

(E) Cell-cycle progression of the yeast samples as in (D) monitored by fluorescence-activated cell sorting (FACS) analysis.

(F) CPT sensitivity of tdp1Δ, wss1Δ, and siz2Δ in the 12geneΔ0HSR multi-transporter mutant genetic background (Chinen et al., 2011). Yeast strains were grown on CPT-containing or control YEPD and imaged 72 h post-plating.