Figure 4.
Mus81-dependent generation of mitomycin C-induced DSBs occurs during S phase. (A) Schematic representation of the experimental protocol. Wild-type and Mus81−/− ES cells were incubated in 5 mM thymidine for 12 h. For the last 6 h of the incubation, mitomycin C was added to a final concentration of 2 μg/ml. Control cells were incubated with thymidine-containing media only. Next, the cells were washed twice with PBS, incubated in fresh medium to allow resumption of replication, and collected at the indicated times. (B) Cell cycle profiles of cells treated as described in panel (A), shown as BrdU incorporation versus PI plots. (C) The relative amount of broken DNA in wild-type and Mus81−/− ES cells treated as described in panel (A) was assessed by PFGE.