Figure 1.—

Sae2 blocks the TM pathway. (A) SAE2 overexpression increases the UV sensitivity of mec1 and rad50S mec1. Yeast strains carrying FLAG-SAE2/CEN (low copy) or FLAG-SAE2/2μ (high copy) were 1/10 serially diluted, spotted onto plates, and left untreated (left) or irradiated with UV (right). (B) Whole-cell extracts were prepared from the strains listed in A by TCA preicipitation and were Western blotted with anti-Flag antibody. Extracts from high-copy transformants were diluted to 1/5. (C and D) SAE2 overexpression influences TEL1- and RAD53- dependent pathways. MMS sensitivity of rad50S mec1 tel1 or rad50S mec1 rad53 cells carrying empty vector or FLAG-SAE2/2μ was determined by spotting experiments (1/5 serial dilution). (E) SAE2 overexpression blocks G₂/M checkpoint. G₁-arrested cells were released into 0.02% MMS and the percentage of binucleated cells was determined. Solid symbols with straight lines indicate MMS-treated samples; wild type with 2μ vector (♦), mec1 with 2μ (▪), rad50S mec1 with 2μ (▴), and rad50S mec1 with SAE2/2μ (×). Open symbols with dashed lines indicate MMS-untreated samples. (F) Telomere length control is not a primary target of Sae2 in regulation of the TM pathway. The strains with either SAE2/CEN or SAE2/2μ were analyzed for telomere length by Southern blot.