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. 2013 Mar;33(6):1175–1187. doi: 10.1128/MCB.01713-12

Fig 7.

Fig 7

Top3 is required for viability of pot1Δ rqh1-hd double mutants with circular chromosomes, and reduction of crossover frequency significantly suppressed the lethality of a pot1Δ rqh1Δ double mutant. (A) Chromosomes of the pot1Δ rqh1-hd double mutant were circularized when cells were incubated in EMM medium. NotI-digested S. pombe chromosomal DNA from the wild type, a pot1Δ mutant, a pot1Δ rqh1-hd double mutant plus pREP41 empty vector, a pot1Δ rqh1-hd double mutant plus pREP41-Top3 vector, and a pot1Δ rqh1-hd double mutant plus pREP41-Top3-Y330F vector (JY746, KTA037, KTA047, KTA044, and KTA045) was analyzed using PFGE at 25°C. Cells were cultivated throughout in EMM medium in the presence of thiamine (30 μg/ml) to suppress Rad51 expression. (B) Expression of Top3-Y330F in the pot1Δ rqh1-hd cells with circular chromosomes is lethal. The pot1Δ rqh1-hd double mutant plus pREP41 empty vector, pot1Δ rqh1-hd double mutant plus pREP41-Top3 vector, and pot1Δ rqh1-hd double mutant plus pREP41-Top3-Y330F vector were streaked on EMM plus adenine and uracil (EMM+AU) to induce the expression of Top3 or Top3-Y330F. (C) Spotting assay of 10-fold serial dilutions of cells. pot1Δ rqh1Δ, pot1Δ rqh1Δ rad51Δ, pot1Δ rqh1Δ rad57Δ, and pot1Δ rqh1Δ sfr1Δ cells expressing Pot1 from plasmid (KTA048, TN040, TN077, and TN076) were plated on YEA and YEA plus 5-fluorodeoxyuridine (YEA+FUDR) at 25°C. The Pot1 plasmid is retained on YEA medium, and cells that lost the plasmid were selected against on YEA plus 50 μM FUDR at 25°C.