SAPK Pathway and Recombination Figure 5. Rad52 phosphorylation upon H2O2 treatment is Sty1 dependent. A) Immunoblot with anti-GFP antibodies of protein extracts from strains rad52YFP and sty1-d rad52YFP synchronized by HU (block) and released into cell cycle in the presence of 100 mM H2O2 (upper panel). Delay in cell cycle progression imposed by the treatment was analyzed by FACS analysis (middle panel) and by scoring cells with 2 nuclei (lower panel). doi:10.1371/journal.pone.0047987.g005 the rate of conversion types (Ade+ His+) in sty1-d cells was significantly diminished. This experiment indicates that Sty1 modulates spontaneous intrachromosomal recombination by promoting one of the mechanisms leading to gene conversion. It also suggests that Sty1 acts on HR also in the absence of exogenous oxidative stress. HR at blocked replication forks is diminished in sty1-d cells The results obtained using UVA and H2O2 suggest that the Sty1 pathway impinges on HR at least in cells undergoing S phase. We took advantage of the RuraR inducible system (Figure 7A) described in Lambert et al. [32] to assess recombination induced by replication fork arrest. This system allows replication fork to be blocked at the RTS1 fork barrier near the ura4+ gene (RuraR substrate) on chromosome 3. Induction of replication fork arrest is obtained by controlled expression of Rtf1 protein that binds RTS1 sequence creating a barrier to fork progression: expression is shut down in medium containing thiamine (OFF) and is induced in medium depleted of thiamine (ON) leading to fork arrest. Restart of arrested replication forks requires HR and, occasionally, fork arrest induced recombination will result in genome rearrange- ments (Figure 7A) [32]. A strain deleted for sty1 and containing the Figure 6. Lack of Sty1 impairs efficient HR. (A) Representation of the substrate used to estimate spontaneous recombination rate [54]. Lower panel shows recombination rate of sty1+ and sty1-d cells. Statistically significant differences are indicates by stars. doi:10.1371/journal.pone.0047987.g006 RuraR substrate and the inducible rtf1+ gene was constructed (RuraR sty1-d). Growth rate of this strain was comparable to the one of RuraR sty1+ (not shown). Survival of RuraR sty1-d strain was compared to the RuraR sty1+ and to RuraR rad52-d by growing cells over night in the absence of thiamine (ON) and spotting serial dilutions on plate containing (OFF) or not (ON) thiamine. The RuraR sty1-d strain behaved as RuraR sty1+ (Figure 7B). Restriction fragment length analysis (RFLA) showed that rearrangements were induced by replication forks arrest (ON) in both sty1+ and sty1-d strains. However, RuraR sty1-d cells had decreased levels of ura4+ marker switch and of acentric chromosome formation if compared to strain of reference. Quantification from three independent experiments indicated a significant two fold decrease in % of HR occurring at blocked replication forks in RuraR sty1-d cells (Figure 7C). The reduced formation of acentric chromosome in RuraR sty1-d cells was confirmed by PFGE where chromosome 3 containing the substrate was identified by hybridizing with rng3 probe (Figure 7D). Thus, in line with the results of the experiment measuring spontaneous recombination rates, HR occurring at blocked replication forks is less efficient in cells with disabled SAPK pathway suggesting that it impinges on HR even in the absence of external oxidative stress. Both the lower spontaneous HR rate and the lower percentage of HR at blocked replication forks are consistent with the suggestion that HR is less performing in sty1-d cells exposed to UVA, where Rad52 foci persist longer. Rad52 is constitutively phosphorylated in a Sty1 dependent manner in cells lacking Rhp51 The results presented above indicate that Sty1 modulates HR even in the absence of exogenous oxidative stress and that Rad52 phosphorylation might be implicated in recombination processes. However, Rad52 phosphorylation was not observed in cells not PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e47987