Figure 1.
MNNG Damage Signaling Coincides with S Phase (A) Cell cycle dependence of MNNG damage signaling. Synchronized HeLaS3 cells were released from a double thymidine block and cultured for varying times in complete medium followed by incubation for 1 hr in serum-free medium in the presence or absence of 10 μM MNNG prior to harvesting. Synchronization was monitored by FACS prior to MNNG treatment. Phosphorylated and nonphosphorylated SMC1 and Chk1 were detected by immunoblotting. “C” is an asynchronous cell population. (B) DNA damage signaling in response to O6-meG adducts. HeLaS3 cells synchronized in S phase (2 hr after release from a double thymidine block) or G2-M-G1 phases (12 hr after release) were incubated in the presence or absence of O6-benzyl-guanine followed by incubation for 2 hr in serum-free medium containing 2 μM MNNG. Lysates were analyzed by immunoblotting.