oxoG), cyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), single strand breaks and DNA protein cross-linking [2]. 8-oxoguanine DNA glycosylase OGG1, which removes 8-oxoG, efficiently prevents UVA-induced muta- genesis in yeast Saccharomyces cerevisiae [30]. HR is a DNA repair pathway dedicated to DSBs repair and recovery of blocked replication forks. Failure to repair DSBs results in cell death while inaccurate repair results in genome instability. HR must be tightly regulated to avoid dangerous outcomes that challenge genome stability and to prevent accumulation of DNA structures that are toxic for the cell [31]. At stalled replication forks HR allows to resume replication following fork collapse, but this mechanism can potentially induce gross chromosomal rearrangements and thus genomic instability [32]. S. pombe encodes a Rad52 homologue called spRad22. Here we will refer to the fission yeast protein as Rad52. This protein, a so- called mediator protein central to HR, is essential for both Rad51 (spRhp51) dependent and independent DSB repair pathways [33,34]. Rad52 is required to replace Replication protein A (RPA) bound to single stranded DNA (ssDNA) by Rad51 (called Rhp51 in fission yeast), which will in turn promote homology search and D-loop formation [35]. However, Rad52 can promote HR in a Rad51 independent way because of its ability to anneal complementary single stranded DNA [36]. Recombination proteins, including Rad52, localize at double strand breaks to form discrete foci that occurs either spontaneously in S phase cells or after induction of DNA damage. In budding and fission yeast, Rad52 foci formation is a marker of ongoing HR [37,38]. We previously reported that, in both mammals and fission yeast, DNA replication is perturbed by UVA exposure and that, despite activation of checkpoint pathways, UVA induced delay in DNA synthesis is largely checkpoint and Sty1 independent [39,40]. In addition, we have shown that cells lacking Sty1 are UVA sensitive, indicating that the SAPK pathway plays an important role in response to this radiation also in fission yeast. Furthermore, cells exposed to UVA during S phase accumulate HR foci and this DNA repair pathway is required for survival to UVA [39]. We sought to further investigate on the interplay between HR and SAPK pathways in response to oxidative stress using UVA and hydrogen peroxide (H2O2). Here we report that Rad52 is phosphorylated in cells exposed to exogenous oxidative stress or in cells lacking Rhp51 and that phosphorylation is dependent on efficient SAPK pathway. Disabling the SAPK pathway delays resolution of UVA-induced Rad52 foci, affects spontaneous HR and HR occurring at blocked replication forks. Materials and Methods Yeast strains, media, growth conditions The strains used in this study are listed in Table 1. Standard techniques were used for yeast growth and strains construction. Strains were grown in YE-rich medium (DIFCO) containing 2% glucose and supplemented with adenine, leucine, uracile, arginine and histidine [41]. Strains containing the RuraR substrate were grown in EMM glutamate (MP Biomedicals) medium with or without thiamine as described in [32]. Genotoxic, oxidative and anti-oxidant treatments Synchronization in early S phase was achieved by 4 hours treatment with 12 mM hydroxyurea (HU) (Sigma). Cells were then collected, resuspended in H20 and UVA irradiated as described in [39]. UVC irradiation was achieved using Strata- SAPK Pathway and Recombination linkerTM (Stratagene) as described in [42]. Camptothecin (CPT) (Sigma) and H202 treatment (Sigma) was performed by releasing HU synchronized cells into fresh medium containing the chemical at the indicated concentrations. Anti-oxidant treatment was achieved adding 30 mM of N-acetylcysteine (NAC) (Sigma). SDS-PAGE, immunoblot and phosphatase treatment Protein extracts were done according to [42]. To analyze Rad52, 80 mg (unless differently stated) of each protein extract was separated by electrophoresis at 40 Volts over-night on 7.5% acrylamide SDS-PAGE (acrylamide : bis-acrylamide 37.5: 1) using the STURDIER vertical SE 400 gel unit (Hoefer Scientific Instruments). Proteins were transferred for 2 hours at 120 Volts on nitrocellulose membrane (PROTRAN Whatman) using a Biorad Trans-BlotH Cell system. Membranes were probed with mouse anti-GFP antibody (Roche). Equal amount of protein extracts were incubated for 1 hour at 30uC with or without l phosphatase (New England Biolabs) according to manufactory instructions. Microscopy and flow cytometry Percentage of cells with Rad52 foci was scored on micropho- tographs as described in [37]. DNA content was analyzed by staining fixed cells with sytox green (Invitrogen) followed by flow cytometry analysis with FACSCalibur flow cytometer (Becton Dickinson). Data were plotted using CellQuest software. Percent- age of cells with 2 nuclei was scored by staining fixed cells with DAPI (49,6-diamidino-2-phenylindole, Sigma). Intracellular peroxide levels were measured as described in [16]. Breafly, 1 ml of exponentially growing cells was incubated with 30 mM DHR123 (Dihydrorhodamine 123, Invitrogen) and 4.4 mM PI (Propidium Iodide, Sigma) for 30 minutes in the dark at 30uC. ROS production (DHR123) and dead cells (PI) were simultaneously analyzed using a FACSCalibur flow cytometer. ROS levels of unstained PI cells (living cells) were normalized to cell size. Fluctuation test and HR rate estimation Fluctuation test was done as follow: 9 Ade- His+ colonies were independently inoculated in 10 ml of YE-rich medium and incubated at 30uC with agitation till cultures were around 56107 cells/ml for reasons explained in the text. Cells were plated on YE-rich medium to estimate viability. About 36105 cells were plated on EMM medium lacking adenine to estimate the frequency of Ade+ recombinants. Plates were replicated on EMM lacking adenine and histidine to estimate frequency of Ade+ His+ recombinants. Frequencies were analyzed by MSS-MLE (Ma- Sandri-Sarkar Maximum Likelihood Estimator) method with the program FALCOR (Fluctuation AnaLysis CalculatOR) to esti- mate the rate of recombination [43]. For each strain at least three independent experiments of nine cultures each were performed. RuraR assay, RFLA and PFGE Procedure for RFLA (Restriction Fragment Length Analysis) to determine % of recombination at blocked replication forks and PFGE (Pulse Field Gel Electrophoresis) are extensively described in [44,45]. Briefly, one single colony of each strain was grown in 10 ml of EMM + thiamine (OFF) for 24 hours at 30uC. Cells are then washed twice and inoculate in EMM + thiamine (OFF) and EMM - thiamine (ON) for 24 hours. Recombination between the RTS1 inverted repeats is detected by Southern blot on genomic DNA digested either with Ase I or with EcorV restriction enzyme using the ura4 probe. % of recombination was estimated on the PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47987