SAPK Pathway and Recombination Figure 2. H2O2, but not UVC radiation or CPT, induces Rad52 phosphorylation. (A) FACS analysis (left panel) of rad52YFP cells synchronized in early S phase by HU (block), collected in water and released into fresh medium either without treatment (untreated), or after exposure to 100 J/m2 of UVC radiation, or in the presence of 20 mM of CPT. 1C and 2C indicate DNA content. Percentage of cells with 2 nuclei scored in untreated and CPT treated cells shows the delay in mitotic entry imposed by the treatment (right panel). Time 0 is immediately after release into cell cycle in presence or not of CPT. Aliquots of cells at different time points were processed for protein extracts that were analyzed by immunoblot with anti-GFP antibodies to detect Rad52 protein. (B) Cell cycle progression and Rad52 phosphorylation at different time points of rad52YFP cells synchronized in early S phase (block) and released into cell cycle in the presence of 250 mM of H2O2. (C) Cell cycle progression and Rad52 detection at different time points of rad52YFP cells synchronized in early S phase (block) and released into cell cycle in the presence of 250 mM of H2O2 and 30 mM of anti-oxidant (+ NAC). doi:10.1371/journal.pone.0047987.g002 cycle slow down and Rad52 phosphorylation, pointing to the possibility that both events rely on ROS. Rad52 phosphorylation is Sty1 dependent We previously showed that checkpoint Chk1 kinase is phosphorylated in S phase cells irradiated with UVA [39]. We asked if Chk1 would be responsible for the Rad52 phosphorylation observed in UVA irradiated cells. Cultures of wild-type (rad52YFP) and chk1 mutant cells (chk1-d rad52YFP) where synchronized by HU in early S phase, exposed to 400 kJ/m2 and release into cell cycle. Rad52 protein was analyzed after HU treatment (block) and at 0 and 90 minutes after release. As shown in Figure 3A, Rad52 phosphorylation was detected in both strains, indicating that Chk1 kinase is not required for Rad52 modification. We questioned if the Cdc2 kinase, a major cell cycle regulator, would be implicated in Rad52 phosphorylation. To do that we took advantage of the thermosensitive allele cdc2-33 [50]. Cells synchronized in early S phase were released into cell cycle after UVA irradiation either at permissive temperature of 25uC, or at non permissive temperature of 37uC. Rad52 phosphorylation was PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e47987