FIG. 1.

Caffeine overrides the S-M replication and G₂-M DNA damage checkpoints. (A) Caffeine overrides the S-M replication checkpoint. A cds1-2HA6HIS (NB2118) strain was arrested in early S phase with 12 mM HU for 3 h prior addition of 10 mM caffeine for the indicated times. Cell division was monitored by counting septated cells. Three hours after addition of caffeine, cells were stained with DAPI to visualize DNA (right). Arrowheads indicate examples of cells that have divided with unequally segregated DNA. (B) The in vivo effect of caffeine is mimicked by a rad3-ts allele. A rad3-ts strain (PS2358) was grown at 25°C and arrested in early S phase with 12 mM HU for 3 h. Half of the culture was then shifted to the restrictive temperature of 35.5°C, while the other half was kept at 25°C. Septation was monitored for 3 h, and DAPI staining was performed as described above. Arrowheads indicate examples of cells that have divided with unequally segregated DNA. (C) Caffeine overrides the G₂-M DNA damage checkpoint. Strain NB2118 was either mock treated or pretreated with 10 mM caffeine 15 min prior to addition of bleomycin (5 mU/ml), a drug that induces DNA damage. Septation was monitored for the next 3 h.