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. 2020 Mar 18;48(8):4274–4297. doi: 10.1093/nar/gkaa103

Figure 4.

Figure 4.

RER-deficient Rnr1-depleted triple mutants bearing Pol ϵ-M644G or δ-L612M, but not α-L868M, show Top1-dependent severe growth defects. (A) RER-deficient mutants depleted of Rnr1 and bearing Pol ϵ-M644G or δ-L612M show severe growth defects, but these defects are suppressed in absence of Top1. Drop test growth assays of strains carrying PGAL:3HA-RNR1 without gene deletion (labeled “none"), or with deletion of the gene TOP1 or RNH201, or both genes TOP1 and RNH201, and strains carrying both PGAL:3HA-RNR1 and allele pol2-M644G, pol3-L612M, or pol1-L868M, without gene deletion (labeled “none"), or with deletion of the gene TOP1 or RNH201, or both genes TOP1 and RNH201, and strains carrying both PGAL:3HA-RNR1 and allele pol2-M644L, without gene deletion (labeled “none"), or with deletion of the gene RNH201. Cells were grown in YPGS (2% galactose and 1% sucrose) liquid medium overnight at 30°C. Serial dilutions were plated on YEPD (2% glucose) and YPGS solid media. Plates were incubated at 30°C. Photographs were taken at the indicated number of days (d). The images for each incubation time are from the same plate. The horizontal lines across the images are included for clarity. ‘Glu’ stands for glucose. ‘Gal + Suc’ stands for galactose plus sucrose. For the ease of comparison, a unique Latin alphabet letter is allocated for each row. See Supplementary Table S1 for the list of strains. One representative experiment is shown of at least three independent ones. (B) The variant Rnh201-RED does not suppress the severe growth defects of RER-deficient mutants depleted of Rnr1 and bearing Pol ϵ-M644G or δ-L612M. Drop test growth assays of strains PGAL:3HA-RNR1 pol2-M644G rnh201Δ and PGAL:3HA-RNR1 pol3-L612M rnh201Δ that have the same plasmids as in Figure 2B. Cells were grown overnight in liquid minimal medium lacking leucine with 2% galactose and 1% sucrose at 30°C. Serial-dilutions were plated on solid minimal medium lacking leucine with 2% glucose, or with 2% galactose and 1% sucrose. Photographs were taken after incubation for 7 days at 30°C. The images for each growth condition are from the same plate. The horizontal line across the images is included for clarity. For other details, see (A).