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. 2011 Jan 10;31(6):1263–1274. doi: 10.1128/MCB.00831-10

FIG. 4.

FIG. 4.

Telomere protection of Est1 depends on its G-quadruplex promotion activity. (A) Growth analysis of est1-D514A and est1-60 mutants in the est1Δ yku80Δ/pRS317-CDC13-EST2 background. A cdc13Δ est1Δ yku80Δ strain carrying both pRS316-EST1 and pRS317-CDC13-EST2 plasmids was obtained from a tetrad dissection and was transformed with pRS315-EST1, pRS315-est1-D514A, pRS315-est1-60, or pRS315 empty vector as indicated. After eviction of pRS316-EST1 plasmid by 5′-FOA selection, two independent clones were sequentially restreaked on selective Leu− plates at 30°C. The number of passages for each streak plate is indicated below. (B) Growth analysis of Est1-D514A overexpression in yku70Δ cells. The plasmid pRS315-yku70, pDBL-EST1 (63), pDBL-est1-D514A or an empty vector was transformed into yku70Δ cells. Five-fold serial dilutions were spotted on Leu− medium and grown at 30 or 37°C as indicated. (C) Growth analysis of the yku70Δ mutant. Plasmid pRS415-CDC13-EST1 (CDC13-EST1), pRS415-CDC13-est1-D514A (CDC13-est1-D514A), or pDBL-CDC13 (CDC13) was transformed into the yku70Δ cells. Five-fold dilutions were spotted on Leu− medium and grown at 30 or 38°C as indicated. The parallel transformation and growth analyses were done with pRS315-YKU70 and pRS415 vector alone for controls. (D) Growth analyses of est2Δ yku80Δ cells in the presence of the CDC13-EST1 or CDC13-est1-D514A fusion gene. est2Δ yku80Δ/pRS316-EST2 cells were generated by tetrad dissection and then transformed with pRS415-CDC13-EST1 or pRS415-CDC13-est1-D514A. The parallel transformation and growth analyses were done with pRS315-YKU80 and pRS415 vector alone for controls. Cells were streaked on the 5′-FOA medium to evict the pRS316-EST2 plasmid. (E) Western blot analyses of Cdc13-Est1 and Cdc13-Est1-D514A expression. pRS415 empty vector, pRS415-Myc-CDC13-EST1, or pRS415-Myc-CDC13-EST1-D514A plasmid was transformed into YPH499 cells, and the total cell lysate was subjected to Western blot analyses with anti-Myc antibody (α-Myc) (upper panel). A nonspecific band shown in the same film was set as a loading control (LC) (lower panel). (F) Growth analysis of the yku70Δ mutant. Plasmid pRS415-CDC13-EST1 was transformed into the yku70Δ or yku70Δ pif1Δ cells. Five-fold dilutions were spotted on Leu− medium and grown at 30, 37, or 38.5°C as indicated. The parallel transformation and growth analyses were done with pRS315-YKU70 and pRS415 vector alone for controls. (G) Telomere Southern blot analysis of est1 mutants in the cdc13Δ yku80Δ est1Δ/pRS317-CDC13-EST2 background. The isogenic strains shown in panel A were restreaked on plates, and genomic DNA was isolated from the 1st, 2nd, 4th, 8th, and 12th restreaks, as labeled at the top of the blot. DNA was digested by XhoI and subjected to Southern blot analysis with a TG1-3 probe. The subtelomeric Y′ signal is indicated on the right.