Fig. 2

Sae2 oligomerization is required to promote the MRX nuclease in vitro and in vivo. a Size exclusion chromatography profile of the C-terminal domain of pSae2 (pSae2 ΔN169, residues 170–345) prepared with phosphatase inhibitors and either mock-treated (pSae2 ΔN169, green) or dephosphorylated with λ phosphatase (pSae2 ΔN169 λ, green dashed). The profile of full-length pSae2 (same as in Fig. 1h) is shown for reference. b Representative nuclease assays with pSae2 ΔN169 treated or not with λ phosphatase and MRX. c Quantitation of assays such as shown in b; error bars, SEM, n ≥ 4. d Coomassie-stained polyacrylamide gel showing pSae2 ΔN169 treated or not with λ phosphatase. e A scheme of the Spo11-oligonucleotide assay. See text for details. f Spo11-oligonucleotide assay performed with wt SAE2 (left), sae2Δ (middle), or wt SAE2 expressed from a centromeric plasmid under the control of its natural promoter and terminator (right). The red triangles mark the long and short Spo11-oligonucleotide species generated in wild-type cells. The open bracket marks MRX-Sae2-independent “double-cut” Spo11-oligonucleotide species observed in wild type and sae2Δ (D.J., V.G., and M.J.N, manuscript in preparation). The black triangle marks a non-specific terminal deoxynucleotidyl transferase band. g–h Spo11-oligonucleotide assay as in f, performed in sae2Δ cells with wt SAE2 or N-terminal truncations expressed from an inducible promoter 4 h after the onset of meiosis. Samples from uninduced cells are labeled in black; samples from cells induced with β-estradiol are labeled in red. The numbers below represent the amount of Spo11-oligonucleotides relative to wild type