Fig. 5.

Mrc1 does not contribute to checkpoint activation after cdc13-1 dependent telomere uncapping. (A and B) 7 cdc13-1 strains, whose genotypes are shown in e, were switched from 23 °C to 27.3 °C and their cell cycle position and growth were monitored, as described in Section 2. (C and D) cdc15-2 bar1 strains with additional mutations [DLY2646 (cdc13-1 mrc1Δ), DLY3071 (cdc13-1 mrc1Δ exo1Δ), DLY1468 (cdc13-1), DLY1470 (cdc13-1 rad9Δ)] were synchronised with α-factor, released from G1 to non-permissive temperature (36 °C) to induce telomere uncapping and cell cycle position was measured, after cells were fixed in 70% ethanol and stained with DAPI. (E) Genotypes of strains used for the asynchronous cultures demonstrated in (a and b) are shown. (F) Western blot demonstrating Rad53 phosphorylation in various cdc13-1 strains with the additional mutations indicated [DLY1108 (cdc13-1 RAD+), DLY1256 (cdc13-1 rad9Δ), DLY2532 (cdc13-1 mrc1Δ), DLY2533 (cdc13-1 mrc1Δ)]. Cultures were grown overnight at 23 °C, diluted in the morning and divided in two. While still growing exponentially, the temperature was raised to 36 °C in one of the aliquots and samples were taken 2 h later and processed for Western blots. Anti-Tubulin was used as a loading control.