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. 2022 Dec 19;42(8):e111500. doi: 10.15252/embj.2022111500

Figure EV2. Double mutants of bir1∆ suppressors in the Dam1 complex and the dam1(S20D) phosphomimic have strong synthetic phenotypes.

Figure EV2

  1. Tetrad dissections performed on diploids with both sli15∆ and the duo1(P17L) did not produce any viable spores with either sli15∆ or a combination of sli15∆ and duo1(P17L).
  2. Cell cycle progression as measured by percentage of large budded cells over time after release from G1 arrest. None of the tested suppressor mutants cause a mitotic delay.
  3. Tetrad dissections of diploids heterozygous for both a bir1∆ suppressor mutant and the dam1(S20D) phosphomimic mutant. Presence of either the suppressor mutant or dam1(S20D) was determined by antibiotic resistance conferred by a gene integrated downstream of the mutations (clonNAT and G418 respectively). Colonies from haploid spores containing either dad2(K11Q), dam1(S20D) or duo1(P17L), dam1(S20D) were extremely rare. Spore viability of each genotype was counted in tetrads whose type (parental ditype, nonparental ditype, or tetratype) could be distinguished. Quantification for each mutant is from a single plate, and the WT numbers are the averages from all six plates. The representative image to the right of the graph depicts six tetrads, demonstrating examples of three tetratypes, two nonparental ditypes, and a single parental ditype. In this example, the expectant duo1(P17L), dam1(S20D) double‐mutant formed a colony from 0 out of seven spores.
  4. Proportion of colonies that grow on plates with restrictive (lacking uracil) versus permissive (YPAD) media after 24 h growth under permissive conditions with a URA3‐containing plasmid. duo1(P17L), dam1(S20D) double mutants have a strong decrease in transmission fidelity. Data are from three independent experiments Means and standard deviations are shown.
  5. Cell cycle progression as measured by percentage of large budded cells over time after release from G1 arrest. Rare surviving duo1(P17L), dam1(S20D) double mutants have a strong mitotic delay. Wild‐type (WT) cells treated with nocodazole and benomyl were used as a positive control for mitotic arrest.
  6. Serial dilution showing growth of strains containing either the double‐mutant (duo1(P17L), dam1(S20D)), the ipl1‐321 temperature‐sensitive allele, or a combination of all three mutations. At the restrictive temperature (34°C), the double‐mutant rescues growth of the ipl1‐321 allele. Ten‐fold serial dilutions were done on YPAD plates at the indicated temperatures and were grown for 48 h.

Data information: (ns) not significant; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; unpaired t‐test.