Fig 1.

pot1Δ rqh1Δ rad51Δ and pot1Δ rqh1Δ exo1Δ triple mutants are viable and lose telomeric DNA. (A) Spotting assay of 10-fold serial dilutions of cells. The pot1Δ, pot1Δ rqh1-hd, pot1Δ rqh1Δ, pot1Δ rqh1Δ rad51Δ, and pot1Δ rqh1Δ exo1Δ cells expressing Pot1 from plasmid (YI002, GT000, KTA023, KTA024, and KTA026) were plated on EMM plus adenine and leucine (EMM+AL) and YEA+FOA at 25°C. The Pot1 plasmid is retained on EMM+AL, and cells that lost the plasmid were selected against on YEA+FOA at 25°C. (B) The telomere lengths of the three independent pot1Δ rqh1Δ rad51Δ and pot1Δ rqh1Δ exo1Δ strains were analyzed using Southern hybridization at 25°C. pot1Δ rqh1Δ rad51Δ cells and pot1Δ rqh1Δ exo1Δ cells that express Pot1 from plasmid (+Pot1p) were used as controls that have telomeric DNA. Genomic DNA was digested with EcoRI and separated by 1.5% agarose gel electrophoresis. A telomere fragment (Telomere) plus telomere-associated sequence (TAS1) derived from pNSU70 (72) was used as a probe. To assess the total amount of DNA, the gel was stained with ethidium bromide (EtBr) before blotting onto the membrane was performed. (C) Restriction enzyme sites around the telomere and TAS1 of 1 chromosome arm cloned in the plasmid pNSU70 (72).