SAPK Pathway and Recombination Figure 7. Sty1 is required for efficient HR occurring at blocked replication forks. (A) Schema representing the RuraR substrate and the possible outcomes after induction of replication block [32]. (B) Drop assay of RuraR sty1+, sty1-d and rad52- d cells grown overnight in the absence of thiamine and spotted on medium containing (OFF) or not (ON) thiamine. (C) RFLA analysis using ura4 probe of recombination outcomes in RuraR sty1+ and RuraR sty1-d cells under conditions of induction (ON) or not (OFF) of replication fork arrest. The % of recombination was estimated from three independent experiments. Stars indicate statistically significant differences. (D) Analysis by PFGE of acentric chromosome 3 formation upon induction of replication fork arrest (ON) using rng3 probe and quantification from two independent experiments. Stars indicate statistically significant differences. doi:10.1371/journal.pone.0047987.g007 exposed to oxidative stress. As we suggest in the discussion, a Sty1 dependent phosphorylation of Rad52 might be difficult to detect in untreated cells because of low basal levels of HR. Thus, we looked at Rad52 phosphorylation in cells lacking the Rhp51 recombinase (rhp51-d rad52YFP) where early steps of HR are impaired [35]. We found that a slower migrating band of Rad52, which can be reversed by lPP treatment, was detected in cells lacking Rhp51 in the absence of any external insult (Figure 8A, first and third lanes), indicating that Rad52 is constitutively phosphorylated when early step of HR is prevented. More importantly, deletion of sty1 gene abolished Rad52 phosphoryla- tion in rhp51-d cells (Figure 8A, second lane). In addition, the intracellular concentration of ROS in rhp51-d rad52YFP cells, measured using the redox sensitive fluorescent probe DHR123, PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e47987