Fig. 3

CDK phosphorylation of Sae2 is prerequisite, but not sufficient, to promote the MRX nuclease. a Nuclease assays with MRX and pSae2 variants mutated in one of the three CDK consensus sites (S267, S134, and S179) into alanine. All mutants were expressed in the presence of phosphatase inhibitors. Substrate used was the same as in Fig. 1c. b Quantitation of the assays such as shown in a; error bars, SEM; n ≥ 3. c Nuclease assays with pSae2 S267E phosphomimicking variant, expressed in the presence of phosphatase inhibitors. The pSae2 S267E protein was mock or λ phosphatase treated, as indicated. d Quantitation of assays such as shown in c; error bars, SEM; n ≥ 3. e Representative polyacrylamide gel (4–15%) electrophoretic analysis of the indicated pSae2 variants, treated or not with λ phosphatase, stained with Coomassie brilliant blue. f Sae2 was expressed and dephosphorylated with λ phosphatase during purification (Sae2 λ). The polypeptide was then mock-treated or phosphorylated with recombinant CDK1/Cyclin B, where indicated, and used in nuclease assays (60, 120, and 200 nM, respectively) with MRX. The pSae2 S267E variant treated with λ phosphatase (pSae2 S267E λ, 200 nM) was used as a reference. g Quantitation of assays such as shown in f; error bars, SEM; n ≥ 3. h Spo11-oligonucleotide assay, as in Fig. 2f, performed in sae2Δ cells with wt, and sae2 mutant bearing non-phosphorylatable (S→A) or phosphomimicking (S→E) mutations in the CDK consensus sites of Sae2 expressed from a centromeric vector. Quantitation is shown below the lanes as an average (relative to wild type) ± coefficient of variation