FIGURE 3.

Caffeine-sensitive checkpoint pathway signaling is important for survival of unchallenged mms21Δsl cells. A, caffeine-sensitive growth of mms21Δsl mutant cells. Wild type (SLY1351) and mms21Δsl (SLY1415) were grown to midlog phase, serially diluted, and spotted onto a YPD agar plate (top left) and YPD agar plates having the indicated concentrations of caffeine. B, caffeine overrides activation of Mrc1 in HU- and MMS-treated cells. HU (150 mm) or MMS (0.03%) was added to asynchronous cultures of strains Mrc1-3HA (SLY1351) and Mrc1-3HA/mms21Δsl (SLY1415) growing at 25 °C in YPD medium. After 2 h of incubation, caffeine (10 mg/ml) was added to one-half of the culture and further incubated for 1 h. Whole cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting using anti-HA antibody. The arrow indicates the location of Mrc1, * indicates location of the replication stress-induced modified Mrc1, and the label B indicates an anti-HA cross-reactive background band present in these cell lysates. The label b indicates a blank lane, and u denotes a lane having lysate from the parental untagged strain. C, caffeine abrogates activation of Rad9 and Rad53 in unchallenged mms21Δsl cells. Rad53-3HA (SLY1100), Rad53-3HA/mms21Δsl (SLY1104), Rad9-3HA (SLY1095), and Rad9-3HA/mms21Δsl (SLY1098) strains were grown to midlog phase, caffeine (10 mg/ml) was added to one-half of the culture, and strains were incubated further for 1 h. Whole cell extracts were resolved by SDS-PAGE and analyzed by Western blotting using the anti-HA antibody. + indicates samples in which caffeine was added, − indicates that no caffeine was added, and * indicates the location of caffeine-sensitive modified bands of Rad9 (left) or Mrc1 (right).