Figure 8. Sty1 dependent Rad52 phosphorylation is constitu- tively detected in cells lacking Rhp51. (A) Immunoblot with anti- GFP antibodies of protein extracts from strains rhp51-d rad52YFP (left lane) and rhp51-d sty1-d rad52YFP (middle lane). The change in mobility shift of Rad52 protein in rhp51-d rad52YFP cells is abolished by treatment with l PP (right lane). (B) Measurement of intracellular ROS levels using the fluorescent probe DHR1,2,3 in rad52YFP and rhp51-d rad52YFP cells. Bar represents the SEM of two independent exper- iments. doi:10.1371/journal.pone.0047987.g008 was similar to the control strain (Figure 8B), indicating that intrinsic oxidative stress is unlikely what is triggering the Sty1 dependent Rad52 phosphorylation in rhp51-d cells. Thus, these results support the notion that Sty1 pathway impinges on HR through phosphorylation of Rad52 even in the absence of treatments inducing oxidative stress. Discussion Here we present evidence that the SAPK pathway phosphor- ylates Rad52, a protein central to HR repair, when cells are subject to oxidative stress either by exposure to UVA or to H2O2. We also show that in the absence of exogenous treatment the Sty1 kinase promote spontaneous mitotic gene conversion and recom- bination induced by replication fork arrest. Importantly, we show that cells lacking the Rhp51 recombinase display constitutive Rad52 phosphorylation that is also dependent on Sty1 kinase. A role for fission yeast Sty1 in HR has been shown for meiotic hotspot recombination where Sty1 regulates in a phosphorylation- independent manner the positioning of transcription factor Atf1 at the M26 hotspot. The Sty1 role in meiotic hot spot recombination together with the observation that it is dispensable for basal meiotic recombination, suggest that Sty1 does not regulate general key elements of recombination during meiosis [55]. Rather, chromatin remodeling in meiosis triggered by Sty1 activation in response to nitrogen starvation allows large protein complexes, such as the HR machinery, to be recruited at the hotspot recombination sites [56]. It has also been proposed that all three major MAPK pathways affect mitotic HR in human malignant glioma, but the molecular basis of the MAPK impact on HR remain unknown, and it was concluded, based on the use of SAPKs inhibitors, that ERK and SAPK Pathway and Recombination JNK positively regulate HR while p38 has an opposite impact on it [57]. In the first part of our work we studied Rad52 modification in replicating cells exposed to different exogenous treatments. Sty1 dependent Rad52 phosphorylation was observed upon UVA and H2O2 treatments in replicating cells. Both agents induce a delay in DNA replication progression, but this is not likely the reason leading to Rad52 phosphorylation, since we show that UVC radiation, which also delays replication, did not induce Rad52 phosphorylation. In addition, induction of replication stress in S phase by CPT that leads to requirement for HR also did not result in Rad52 phosphorylation, suggesting that collapse of replication forks because of replisome loss is not a requirement for Rad52 modification. Rather, Rad52 phosphorylation correlated with ROS production since concomitant treatment of cells with oxidant and antioxidant reduced the replication delay and Rad52 phosphorylation. However, Rad52 phosphorylation is not re- quired for replication delay upon oxidative stress, since cells lacking Sty1, and thus Rad52 phosphorylation, still delay replication (this work and [39]). These observations suggest that the Sty1 dependent phosphor- ylation of Rad52 is induced by oxidative stress rather than by DNA lesions. Though, we didn’t observed Rad52 phosphorylation after UVC although it has been shown that UVC radiation induces the Sty1 oxidative stress response explaining the hyper- sensitivity to UVC of sty1-d cells [58]. This discrepancy might outline the greater requirement for HR repair after UVA and H2O2 than after UVC radiation or might reflect different responses to different levels of oxidative stress. According to our results, the response to oxidative stress is required to promote HR since Rad52 foci persist longer in cells lacking Sty1 where Rad52 is not phosphorylated. We do not know if Rad52 is a direct substrate of Sty1 and we cannot exclude that another kinase acting downstream of Sty1 phosphorylates Rad52 in the nucleus. Although we didn’t succeed in identifying by 2- dimensional electrophoresis followed by mass spectrometry the sites of phosphorylation because the Rad52YFP protein failed to separate by isoelectric focusing under different experimental conditions, we ruled out the contribution of kinases Chk1, Cdc2, Rad3 and Tel1 in Rad52 phosphorylation. We have shown that the rate of spontaneous HR is reduced in sty1-d cells that are not exposed to exogenous oxidative stress, and that loss of recombinants is at charge of gene conversion types. Differently from meiotic recombination, mitotic HR is rarely associated with crossovers and different published data suggest that SDSA (Synthesis-Dependent Strand Annealing) is the major non- crossovers mechanism in DSBs repair during mitosis. In SDSA as well as in DSBR (DSB Repair) model, after resection at DSB, a 39 single strand DNA tail is generated that will be coated by Rad51 through a process requiring Rad52. This permits strand invasion and D loop formation allowing DNA synthesis using the homologous unbroken DNA sequence as a template. In SDSA, differently from DSBR, the D loop is displaced and newly synthesized DNA pairs to the other end of DSB. If the homologous sequences are heteroallelic, then the paired DNA will contain a mismatch. Alternatively, a mismatch between the invading strand and the unbroken molecule is formed during D loop extension. Mismatches are detected by the mismatch repair system (MMR) that will either reject the invading strand or correct the mismatch possibly leading to gene conversion. Similarly, the recombination dependent genome rearrange- ments upon replication fork arrest in the RuraR system result from a mechanism of template exchange where the newly synthesized strand at blocked replication fork will engage in formation of D PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e47987