Figure 3. Rad52 phosphorylation upon UVA is independent of Chk1, Cdc2, Rad3 and Tel1 kinases. (A) Cells expressing or not the Chk1 kinase were irradiated with either 0 or 400 kJ/m2 UVA after synchronization in early S phase and Rad52 protein was detected by immunoblot with anti-GFP antibodies at 0 and 90 minutes after release. (B) Rad52 protein was detected at the indicated time points in the cdc2- 33ts strain synchronized by HU treatment at permissive temperature (25uC) and released into cell cycle upon UVA radiation either at permissive or non permissive (37uC) temperature. (C) Rad52 protein detected in rad3-d asynchronous cells after exposition (+) or not (2) to UVA radiation and in tel1-d cells synchronized by HU treatment (block) and released into cell cycle for 0 and 90 minutes after treatment with either 0 or 400 kJ/m2 UVA. doi:10.1371/journal.pone.0047987.g003 assessed immediately after release (time 0) and after 90 minutes (Figure 3B). At both temperatures Rad52 phosphorylation was detected indicating that Cdc2 is unlikely the kinase required for such modification. We then asked if Rad3 (ATR), the main upstream kinase in DNA damage checkpoint, or Tel1 (ATM) kinases were involved in Rad52 phosphorylation. Because cells lacking Rad3 (rad3-d) cannot be synchronized by HU in early S phase, we used asynchronous cultures to assess the phosphorylation state of Rad52 in this genetic background. Differently, cells lacking Tel1 (tel1-d) were synchronized by HU and released into cell cycle after exposure to either 0 or 400 kJ/m2 UVA. We found that upon UVA exposure Rad52 phosphorylation is still detected in both rad3-d and tel1-d cells (Figure 3C). Because UVA treated cells experience oxidative stress that elicits activation of the SAPK pathway, we asked if Sty1 kinase would be involved in Rad52 phosphorylation upon UVA. At first, we compare Rad52 expression from exponentially growing cells expressing or not Sty1 (sty1+ rad52YFP and sty1-d rad52YFP strains) (Figure 4A) and found no differences between the two strains. Then we analyzed Rad52 in sty1-d rad52YFP cells exposed to UVA radiation. Rad52 protein was detected prior HU addition (asyn), after 4 hours of HU treatment (block), and every 30 minutes from time 0 till 120 minutes after irradiation. As a control Rad52 protein from rad52YFP strain was analyzed at 0 and 90 minutes SAPK Pathway and Recombination from release after UVA exposure. As shown in Figure 4B, Rad52 phosphorylation was not detected in UVA-treated cells lacking Sty1, although cell cycle progression was clearly delayed by radiations as judged by the delay in the appearance of cells that have passed mitosis. Because S phase cells exposed to UVA radiation accumulate Rad52 foci in a dose dependent manner [39] and we showed that Rad52 phosphorylation is Sty1 dependent, we asked if the kinetics of HR foci formation would be affected in sty1-d cells. To determine the % of cells having Rad52 foci, rad52YFP and sty1-d rad52YFP cells were synchronized in early S phase, exposed or not to 200 kJ/m2 of UVA and then released into cell cycle. Percentage of cells with Rad52 foci was scored at different time points. Rad52 foci were induced by UVA in both strains, however they persisted longer in sty1-d rad52YFP cells if compared to rad52YFP strain (Figure 4C). At 180 minutes, about 85% of cells lacking Sty1 had Rad52 foci, in contrast to 55% in control strain (Figure 4C). It is to note that the % of cells with Rad52 foci in sty1-d cells released from the HU block without exposure to UVA radiation is greater than in rad52YFP cells up to 60 minutes, but then reaches wt levels suggesting that the first round of replication after HU is perturbed in cells lacking Sty1. In addition, as shown in figure 4D, deletion of Sty1 also abolished Rad52 phosphorylation in asynchronous cells exposed to UVA and lacking Rad3 kinase (rad3-d sty1-d rad52YFP). It has been shown in fission yeast that the stress responsive protein Srr2 is phosphorylated in a Sty1 dependent manner upon stress, resulting in nuclear translocation and binding to Rad4 (TopBP1) [51], a scaffold protein that plays a role in both DNA replication and checkpoint response [52]. Thus, we asked if Srr2 might be required for the Sty1 dependent phosphorylation of Rad52 upon UVA. To answer this question we constructed a strain deleted for srr2 gene and expressing Rad52YFP protein (srr2-d rad52YFP). This strain was synchronized in early S phase, irradiated or not with UVA and released into cell cycle. Immediately (0) and at 90 minutes after release, the Rad52 phosphorylation was examined. As shown in Figure 4E, absence of Srr2 protein did not prevent Rad52 phosphorylation. Then we asked if phosphorylation of Rad52 is Sty1 dependent in cells exposed to H2O2. Treating sty1-d cells with 250 mM of oxidant repetitively resulted in almost undetectable Rad52 protein (data not shown), likely due to the Sty1 requirement to support protein synthesis upon exposure to H2O2 [53]. Thus, we assessed Rad52 phosphorylation upon exposure to lower concentration of oxidant (100 mM) in both Sty1 proficient and deficient cells. 80 and 120 mg of protein extracts were analyzed for rad52YFP and sty1-d rad52YFP cells, respectively. As shown in Figure 5, Rad52 modification was detected in rad52YFP but not in sty1-d cells although the treatment with 100 mM H2O2 delayed cell cycle progression in both strains as judged by FACS analysis and by scoring the percentage of cells that passed mitosis (cells with 2 nuclei). In conclusion, Rad52 phosphorylation in cells exposed to either UVA or H2O2 is Sty1 dependent. Lack of Sty1 affects spontaneous rate of gene conversion One possibility explaining the persistence of Rad52 foci in sty1-d cells is that HR is less performing in cells lacking the Sty1 kinase. Thus, we assessed the rate of spontaneous HR by fluctuation test in wt and sty1-d cells using the system described in Osman et al. [54]. This system allows measuring the frequency of recombina- tion between non-tandem hetero-allelic duplications of the ade6 gene separated by a region of DNA carrying the his3+ gene. With this substrate two classes of Ade+ recombinants can be PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e47987