Table 1. Strains used in this study. STRAIN rad52YFP chk1-d rad52YFP cdc2-33ts rad52YFP rad3-d rad52YFP tel1-d rad52YFP sty1-d rad52YFP srr2-d rad52YFP sty1+ (MCW429) sty1-d RuraR sty1+ RuraR rad52-d RuraR sty1-d rhp51-d rad52YFP rhp51-d sty1-d rad52YFP doi:10.1371/journal.pone.0047987.t001 GENOTYPE h- rad22YFP:KanR h- rad22YFP:KanR chk1::ura4+ ura4-D18 ade6-M216 h- rad22YFP:KanR cdc2-3ts ura4-D18 h+ rad3::ura4+ rad22:YFP:KanR leu1-32 ura4-D18 h- tel1::KanR rad22:YFP:KanR leu1-32 ura4-D18 h+ rad22YFP:KanR sty1::ura4+ ura4-D18 leu1-32 ade6-M216 h- rad22YFP:kanR srr2::kanR h+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6- M375 int:: pUC8/his3+/ade6- L469 h+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/ade6-M375 sty1::KanR h+ sup35::nmt41::rtf1 RuraR ade6-704 leu1-32 h+ nmt41::rtf1:: sup35 ade6-704 leu1-32 RuraR rad22::KanR h+ nmt41::rtf1:: sup35 ade6-704 leu1-32 RuraR sty1::NatR h+ rhp51::ura4+ ura4-D18 rad22YFP:kanR h+ rhp51::ura4+ sty::ura4+ ura4-D18 rad22YFP:KanR leu1-32 SAPK Pathway and Recombination SOURCE [37] This study This study This study This study This study This study This study [34] [32] [32] This study This study This study basis of three independent experiments. Analysis of acentric chromosome III by PFGE was done using rng3 probe. Thus, this experiment shows that Rad52 is phosphorylated in response to UVA radiation. Results Rad52 is phosphorylated in cells undergoing S phase upon UVA exposure We have previously shown that cells irradiated with UVA in early S phase delay DNA replication and accumulate Rad52 foci, a marker of HR, in a dose dependent manner. We also showed that Rad52 is required for survival of cells irradiated in S phase, indicating that HR is necessary to repair some of the DNA lesions induced by UVA radiations [39]. We took advantage of the strain rad52YFP, which expresses the Rad52 protein tagged in C-terminal with YFP (Yellow Fuorescent Protein) [37], to analyze potential post-translational modifications of Rad52 protein by immunoblot with anti-GFP antibodies in cells exposed to UVA in early S phase. This strain was previously shown to delay DNA replication upon UVA exposure in early S phase similarly to wt cells [39]. Cells were synchronized in early S phase by HU treatment and 2 released into cell cycle after UVA irradiation at 0 or 400 kJ/m . Cell cycle progression was followed by FACS analysis and by staining cells with DAPI in order to monitor the number of cells that have passed mitosis (cells with 2 nuclei). Protein extracts were analyzed from cells prior synchronization (asyn), after 4 hours of HU treatment (block), and every 30 minutes after release into cell cycle from time 0 (immediately after irradiation) till 240 minutes. In non irradiated cells a single band corresponding to Rad52-YFP (referred to as Rad52) was detected at all time points, while in irradiated cells, an additional Rad52 slower migrating band was detected after 60 minutes from release (Figure 1A upper panel), time at which cells are progressing through S phase as shown by FACS analysis (Figure 1A middle panel) and haven’t yet passed into mitosis (Figure 1A lower panel). It is to note that Rad52 protein levels seem to increase in irradiated cells. Treatment with l phosphatase (l PP) of a protein sample from UVA irradiated cells abolished the slower migrating band, indicating that it results from phosphorylation (Figure 1B). Rad52 phosphorylation is observed upon oxidative stress, but not after UVC or CPT treatment In order to establish if Rad52 phosphorylation is specific to UVA treatment, we examined it in S phase cells exposed to different DNA damaging agents such as UVC radiation and CPT. UVC radiation directly damages DNA producing mainly CPDs that block replication fork progression [46]. Thus, we analyzed the Rad52 protein in cells synchronized in early S phase and exposed to 100 J/m2 UVC radiation prior release into cell cycle. DNA replication progression was followed every 30 minutes by FACS analysis. Time 0 corresponds to the moment where cells were released into cell cycle immediately after irradiation. As expected, DNA replication was delayed in UVC treated cells if compared to untreated ones (Figure 2A). However, despite delaying S phase, UVC irradiated cells did not display the slower migrating form of Rad52 indicating that UVC radiation does not induce detectable Rad52 phosphorylation (Figure 2A). CPT is a topoisomerase I inhibitor that, at the concentration of 20 mM, generates DSBs in S phase cells and formation of Rad52 foci. Indeed, similarly to UVA [39], HR is required for cell survival to CPT treatment and cells activate Chk1 kinase to delay G2/M transition [47,48,49]. Thus, we analyzed S phase progression and Rad52 phosphorylation in HU synchronized cells released into cell cycle in the presence of 20 mM CPT. Under these conditions and in contrast to UVA and UVC, S phase progression was not delayed as judged by FACS analysis (Figure 2A) but, as expected, entry into mitosis was delayed by the treatment as judged by the persistence of cells with one nucleus in CPT treated culture (Figure 2A). However, similarly to UVC exposure, Rad52 phosphorylation was not detected (Figure 2A). Then, we asked if an oxidative agent different from UVA would induce replication delay and Rad52 phosphorylation, and if both would depend on the presence of ROS. Thus, cells synchronized in early S phase were released into cell cycle in the presence of 250 mM of H2O2 or in the presence of H2O2 and 30 mM of NAC, an anti-oxidant able to counterbalance H2O2 effects. Cell cycle progression and Rad52 phosphorylation were assessed at PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e47987