FIGURE 4.

In the absence of MMS, Rtt107 was phosphorylated in mutants expressing a compromised SMC5/6 complex. A, cells expressing Rtt107-FLAG were untreated or treated with 0.03% MMS for 1 h. Analytical-scale immunoprecipitations of Rtt107-FLAG were performed and analyzed by immunoblotting with anti-FLAG antibodies. The reduced mobility of Rtt107-FLAG indicated phosphorylation of the protein. Cross-reaction bands were used as a loading control. B, as in A, except immunoprecipitates were left untreated or incubated with λ-phosphatase (λ-PP; 200 units) for 30 min at 30 °C in the presence or absence of EDTA (100 mm). C, cells expressing Rtt107-FLAG with and without MEC1 were treated as in A. All strains contained sml1Δ to suppress the lethality of mec1Δ mutants.