Fig 2. Requirement of Rap1 on chromosome truncation at TG repeats.

(A) Effect of rap1-degron mutation on colony formation in the presence of CuSO₄. Wild-type or rap1-degron mutant (rap1-(Δ)) cells were serially diluted (10-fold) and spotted on medium containing 0, 0.05 or 0.5 mM CuSO₄. (B) Effect of CuSO₄ concentration on Rap1-(Δ) protein expression. Wild-type or rap1-(Δ) cells were initially grown in the absence of CuSO₄ and then incubated with the indicated concentrations of CuSO₄ for 6 hr. Aliquots of cells were collected and subjected to immunoblotting analysis with anti-Rap1 antibodies. (C) Effect of Rap1 depletion on URA3 ^Kl marker loss. Wild-type or rap1-(Δ) cells containing the TG₂₅₀ cassette were first maintained in medium selective for URA3 and then transferred to non-selective medium containing 0.05 mM CuSO₄. Saturated cultures were diluted and spread on 5-FOA plates to determine the rate of URA3 ^Kl marker loss. URA3 ^Kl maker loss was determined as in Fig 1C.