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. 2021 Jul 14;10:e67268. doi: 10.7554/eLife.67268

Figure 3. Mutations in SCC2 and histone genes also suppress scc4Δ lethality.

(A) Tetrad dissection of diploid strains containing SCC4/scc4Δ leu2/scc4-4 ΗTΑ1/hta1R31I. Spores in which scc4Δ is rescued by hta1R31I are circled in blue. (B) Structure of the yeast nucleosome (PDB: 1ID3; White et al., 2001). H2A is shown in blue and H2B in green. Suppressor mutations are shown in yellow. (C) Average calibrated ChIP-seq profiles of Scc1-PK6 in hta1R31I, scc4-4, and hta1R31I scc4-4 cells 60 kb either side of CDEIII plotted as a percentage of the average number of reads obtained for wild-type (W)T cells. Cells were pheromone arrested in G1 at 25°C before release at 35.5°C into medium containing nocodazole. Samples were taken 60 min after release (K22005, K24574, K24568, K22001).

Figure 3.

Figure 3—figure supplement 1. Scc4 helps overcome inhibition of loading by nucleosomes.

Figure 3—figure supplement 1.

(A) Cell cycle progression as measured by FACS of wild-type (WT) and sth1-3 cells arrested in G1 with pheromone prior to release into nocodazole containing medium at 37°C (K23997, K22005). (B) Fraction of cells with buds of cells treated as described in (A). (C) Western blot to measure the levels of Scc1-PK6 and acetylation of Smc3 of cells treated as described in (A). (D) Average calibrated ChIP-seq profile of Scc1-PK6 10 kb either side of CDEIII at 75 min and 105 min after release described in (A). The occupancy ratios (OR) were derived as described in Hu et al., 2015. (E) ChIP-seq profiles of Scc1-PK6 as in (D) at individual loci. Sequences measured in Lopez-Serra et al., 2014 are shaded in orange. (F) Average calibrated ChIP-seq profile of Scc1-PK6 in sth1-3 cells at 105 min after release 60 kb either side of CDEIII plotted as a percentage of the average number of reads obtained for WT cells at either 75 or 105 min after release. (G) Average calibrated ChIP-seq profiles of Scc1-PK6 10 kb either side of CDEIII of cells expressing SMC1 or smc1D588Y in the presence of STH1 or sth1-3. Cells were pheromone arrested in G1 at 25°C prior to release into nocodazole containing medium at 37°C. Samples were taken at 75 min and 105 min post release, and samples at similar cell cycle stages were compared (K22005, K22009, K23997, K24031).