SAPK Pathway and Recombination Figure 4. Rad52 phosphorylation upon UVA is Sty1 dependent, but Srr2 independent. (A) Rad52 expression detected by anti-GFP antibodies in cells proficient or not for Sty1. Stars indicate aspecific bands serving as loading control. (B) Protein extracts at the indicated time points were prepared from cells lacking or not Sty1 and probed with anti-GFP antibodies to detect Rad52 protein (upper panel). Cells were synchronized in early S phase by HU (block) and released into cell cycle after irradiation with either 0 or 400 kJ/m2. ‘‘asyn’’ indicates cells in log phase. Delay in cell cycle progression upon UVA was monitored by scoring the percentage of cells with 2 nuclei (lower panel). Time 0 is immediately after irradiation. (C) % of cells with Rad52 foci monitored in the indicated strains released from the HU block and exposed or not to UVA radiation. Time 0 is immediately after irradiation. (D) Immunoblot with anti-GFP antibodies of protein extracts from asynchronous cells of the indicated strains exposed (+) or not (2) to UVA. (E) Immunoblot with anti-GFP antibodies of protein extracts from cells depleted of Srr2 responsive element upon UVA irradiation in early S phase. doi:10.1371/journal.pone.0047987.g004 distinguished: Ade+ His+ resulting from gene conversion and Ade+ His- resulting from deletion of the his3+ gene located between the two ade6 alleles (Figure 6A). Because sty1-d cells loose viability when reaching saturation in medium containing standard glucose concentrations, HR frequencies were estimated in cultures not yet in stationary phase and used to calculate the rate of spontaneous HR using the MSS-MLE method [43]. As shown in Figure 6B, a significant two fold decrease in the rate of spontaneous HR was unveiled in sty1-d cells. While the rate of deletion types (Ade+ His2) was comparable to that of sty1+ cells, PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e47987