Figure 4. The Rad60∶Ubc9 complex and the Nse2 SUMO E3 ligase are essential to protect tdp1 Δ cells from Top1-induced DNA damage.

(A) A representative tetrad dissection is shown from a cross between rad60^E380R and top1Δ tdp1Δ double mutant cells. The key depicts the genotypes present, which are denoted by various shapes placed around each colony. Wildtype cells do not have a shape placed around them. (B) Serial dilutions of the indicated strains expressing Top1 under a thiamine repressible promoter were spotted onto control or CPT containing media with (+B1) or without (-B1) thiamine to repress or induce Top1 expression, respectively. All strains were incubated at 32°C. (C) ChIP-qPCR assays of an nmt41-inducible Top1-FLAG in the indicated strains at the subtelomeres of Chr 2 (telo2R), the centromeric inner repeats of Chr 2 (cnt2), the rDNA (rDNA2), and upstream of mes1 on Chr 1. The data represents the average DNA recovery compared to the input DNA samples with standard deviations from at least three independent experiments. ChIP-qPCR data of nmt41-Top1-FLAG rad60^E380R tdp1Δ grown in repressed media (+B1) is shown as a negative control. Cells were grown at 25°C. (D) ChIP qPCR assays of the indicated strains as in (C). (E) Cells of the indicated genotype were restruck directly from tetrad dissection plates onto YES media. Cells were incubated at 32°C.