Fig. 4

Mec1/Tel1 phosphorylation of Sae2 stimulates, but is not essential, for its capacity to promote the MRX nuclease. a Nuclease assays with MRX and pSae2 mutated simultaneously at SQ/TQ sites at positions 73, 90, 249, and 279, preventing phosphorylation due to alanine substitutions. The pSae2 mutant was prepared with phosphatase inhibitors. Substrate used was the same as in Fig. 1c. b Nuclease assays with MRX and pSae2 mutated simultaneously at SQ/TQ sites at positions 73, 90, 249, and 279 mimicking phosphorylation due to glutamic acid substitutions. The pSae2 mutant was prepared with phosphatase inhibitors and then treated or mock-treated with λ phosphatase. c Assays as in b, with a pSae2 variant carrying in addition a phosphomimicking mutation at the CDK site of S267. d Quantitation of assays such as shown in a, b; error bars, SEM; n ≥ 3. e Quantitation of assays such as shown in c; error bars, SEM; n ≥ 3. f Representative polyacrylamide gel (4–15%) electrophoretic analysis of the indicated pSae2 variants, treated or not with λ phosphatase, stained with Coomassie brilliant blue. g–j Spo11-oligonucleotide assay, as in Fig. 2f–h, with sae2Δ cells bearing a centromeric plasmid expressing g–j individual non-phosphorylatable (S→A) or h–j phosphomimicking mutations in the SQ/TQ sites of Sae2 at positions 73, 90, 249, 279, and 289, or i with sae2 S278A T279A variant expressed upon addition of β-estradiol 4 h after the onset of meiosis. Quantitation is shown below the lanes as an average (relative to wild type) ± coefficient of variation. k Nuclease assay with pSae2 S289A, pSae2 S289E, and pSae2 S289D mutants and MRX. All pSae2 variants were expressed and purified in the presence of phosphatase inhibitors. l Quantitation of assays such as shown in k; error bars, SEM; n ≥ 3. m Nuclease assay with pSae2 S289E S267E double mutant prepared in the presence of phosphatase inhibitors. The Sae2 variant was then mock-treated or dephosphorylated with λ phosphatase