3 mb pp1

Fission yeast growth, gene targeting, and mating were performed as described (Bamps et al., 2004 (link); Fersht et al., 2007 (link)). TAP purification was performed as described (Guiguen et al., 2007 (link)). TAP immunoprecipitation and kinase assay on GST-CTD were previously described (Drogat et al., 2012 (link)). Inhibitors (1-Nm-PP1 and 3-Mb-PP1) of the analogue-sensitive mutant kinases were purchased from Toronto Research Chemicals (Toronto, Canada). Trichostatin A was purchased from Millipore (Billerica, MA) and used at 50 μg/ml. GST-fusion proteins were expressed and purified using the GE kit according to the manufacturer instructions with the following variations: growth was performed at 18°C and induction performed at 0.5 mg/ml IPTG. The expression of ste11 was induced by nitrogen starvation or by the addition of methionine, as described (Schweingruber et al., 1998 (link); Coudreuse et al., 2010 (link)). The expression of inv1 was induced by shifting cells grown in YE 2% glucose medium to YE 0.1% glucose/glycerol 3% for 1 hr. Western blot were performed with anti-GST (Sigma), peroxidase–antiperoxidase (PAP) (Sigma), anti-S2P (3E10, Covance), anti-S5P (H15, Covance) antibodies.
Phosphatase (λ-phosphatase, New England Biolabs) treatment was performed as described. Iodine staining was performed as described (Bauer et al., 2012 (link)).

Meiotic induction and DNA preparation were performed as described (51 ). Briefly, strains with pat1-114 (temperature-sensitive) or pat1-as1 (ATP analog-sensitive) were used to induce synchronous meiosis after nitrogen starvation (which produces G1-arrested cells) by shifting the temperature to 34°C for pat1-114, or by adding 3-MB-PP1 (Toronto Research Chemicals) to 25 μM at 25°C or 34°C for pat1-as1, and adding NH₄Cl as a nitrogen source. All DNA analyses were done with cultures induced at 34°C except for those in Figure 4B at 25°C. Cells were harvested at the specified times after induction, embedded in agarose plugs, and digested with lytic enzymes to break the cells. The plugs were further treated with proteinase K, subsequently inactivated with phenylmethylsulfonyl fluoride (PMSF), and washed thoroughly with TE buffer.
Induction of meiosis was confirmed by flow cytometry to determine pre-meiotic DNA replication, which typically began at 2 h and was completed by 3 h at 34°C (3.5 and 5.5 h, respectively, at 25°C).