SAPK Pathway and Recombination Figure 1. Rad52 is phosphorylated in S phase cells exposed to UVA radiations. (A) Protein extracts analyzed by immunoblot with anti-GFP antibodies (upper panel) to detect Rad52 protein from rad52YFP cells synchronized in early S phase by HU (block), collected in water and released in fresh medium after 0 or 400 kJ/m2 UVA for different time points. ‘‘asyn’’ indicates cells in log phase. Star refers to the same membrane colored with Rouge Ponceau to serve as loading control. An aliquot of cells at different time points was used to estimate cell cycle progression by FACS analysis and by scoring the percentage of cells that passed mitosis (cells with 2 nuclei). 1C and 2C indicate DNA content. (B) The change in mobility shift of Rad52 protein upon UVA is abolished by treatment with l phosphatase (PP). doi:10.1371/journal.pone.0047987.g001 different time points by FACS analysis and western blot, respectively. In cells exposed to H2O2 DNA replication was delayed (compare FACS panel in figure 2B to FACS panel ‘‘untreated’’ in figure 2A) and Rad52 protein was phosphorylated starting at 30 minutes from release (Figure 2B). When cells were released into cell cycle in presence of both oxidant and NAC, cell cycle delay was diminished if compared to cell exposed only to H2O2, although it was not completely abolished. Indeed, in cells treated with H2O2 the first round of replication ended around 150 minutes post release (Figure 2B), in the presence of H2O2 and NAC it was completed at 90 minutes (Figure 2C), while in untreated cells DNA replication ended around 60 minutes (Figure 2A ‘‘untreated’’). Furthermore, Rad52 phosphorylation was barely visible in cells exposed to H2O2 along with NAC (Figure 2C). In conclusion, UVC and CPT did not induce Rad52 phosphorylation, while treatment with 250 mM H2O2 resulted in DNA replication slow down and Rad52 modification, as for UVA radiation. Furthermore, NAC prevented in part both cell PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47987