Red1 binds Smt3 chains F-M Lin et al In conclusion, this work demonstrates that Red1 protein can bind Smt3 chains. This Red1–Smt3 chain interaction provides a new molecular clue to the role of Red1 in the coordination of homologous recombination, Mec1–Tel1 checkpoint kinase activation and chromosome synapsis during yeast meiosis. Materials and methods Yeast strains and two-hybrid assay Almost all meiotic experiments were carried out using diploid cells on the SK1 strain background. BR strains were kindly provided by Shirleen Roeder (Yale University). Yeast two-hybrid assays were carried out as previously described (Cheng et al, 2006). Cytology, western blot and physical analysis Cytological analyses were carried out as previously described (Cheng et al, 2006). For western blot time-course studies, TCLs were prepared according to the TCA precipitation protocol described previously (Cheng et al, 2006). This protocol uses both TCA and urea (8 M) in the sample buffers, which allows for efﬁcient extraction of proteins from yeast cells grown on various media and the prevention of proteolysis, dephosphorylation and desu- moylation. Physical analyses were performed as described pre- viously (Clyne et al, 2003). Quantiﬁcation of the DSB, NCO and CO signals was achieved using a Fujiﬁlm image analysis program (Image Gauge 4.0). Expression and puriﬁcation of the wild-type and mutant GST–Red1–His6 fusion proteins The DNA fragment encoding wild-type Red1 protein was cloned into a GST–His6 expression vector using the EcoRI and XhoI restriction sites. The GST–Red1–His6 expression vector was then subjected to site-directed mutagenesis to create the GST–Red1I758R–His6 expression vector. The expression vector was transformed into BL21(DE3) competent cells. For each protein, cell culture (100 ml) was grown overnight at 371C with 50 mg/l ampicillin. After transferring the cell culture to 4 l of LB medium, the cell suspension was allowed to reach an OD600 of approximately 0.5–0.6 before 1 mM IPTG was added. Cells were grown for 15 h at 181C and centrifuged at 6000 g for 30 min. The cell pellet was resuspended in 100 ml lysis buffer (50 mM Tris–HCl pH 8.0, 300 mM NaCl, 50 mM sucrose and 0.2 mM EGTA) and disrupted using a French press (30 000 psi; Sim-Aminco). The TCL was centrifuged at 61000 g for 45 min. The soluble protein fraction was loaded onto a 4 ml Ni2 þ -chelating Sepharose column (GE Healthcare). After the column was washed with 50 ml lysis buffer plus 5 mM imidazole (pH 8.0), the His6-tagged proteins were eluted with 8 ml lysis buffer plus 100 mM imidazole. The eluants were then directly mixed with 4 ml glutathione resins (GE Healthcare) to purify the GST-tagged proteins. The full-length GST–Red1–His6 and GST–Red1I758R–His6 proteins were then eluted with 8 ml lysis buffer containing 10 mM glutathione in the presence of 10 mM b-mercaptoethanol. The eluants were collected and dialyzed against buffer Q (50 mM Tris–HCl pH 8.0, 200 mM NaCl, 40% glycerol and 1 mM b-mercaptoethanol). Production of Smt3 chains Smt3 chains were synthesised as previously described (Cheng et al, 2006). Brieﬂy, puriﬁed His6–Myc–Smt3, His6–Aos1/His6–Uba2 complex (E1), His6–Ubc9 (E2), Mg2 þ and ATP were incubated. The resulting products could not be directly applied to the Red1 binding assay because His6–Ubc9 (B18 kDa) and the His6–Ubc9/ References Agarwal S, Roeder GS (2000) Zip3 provides a link between recom- bination enzymes and synaptonemal complex proteins. Cell 102: 245–255 Anderson CM, Korkin D, Smith DL, Makovets S, Seidel JJ, Sali A, Blackburn EH (2008) Tel2 mediates activation and localization of ATM/Tel1 kinase to a double-strand break. Genes Dev 22: 854–859 His6–Myc–Smt3 conjugated adducts (B35 kDa) catalyze the cova- lent addition of His6–Myc–Smt3 to Red1 recombinant proteins. To partially purify the Smt3 chains, gel ﬁltration was carried out by high-pressure liquid chromatography using a HiLoad 16/60 Super- dex sizing column (GE Healthcare) run at a ﬂow rate of 1.0 ml/min. The elution proﬁle was followed by a continuous assay of the optical density at 280 nm. Commercially supplied molecular mass standards, including thyroglobulin, ferritin, catalase, bovine serum albumin (BSA), ovalbumin and lysozyme, were used to calibrate the column. The average elution position, Kav, was computed by the equation Kav ¼ (Ve V0)/(Vt V0), where Ve and Vt represent elution volumes for the molecular mass standards and DTT, respectively. The void volume, V0, was determined using blue dextran 2000 (GE Healthcare). The Smt3 chains were eluted before the elution volume expected for a globular protein with an apparent molecular weight of 60 kDa. The eluants were then analysed by Western blotting using either anti-Myc or anti-Ubc9 antibody (Santa Cruz Biotechnology). The puriﬁed Smt3 chain products contained neither His6–Myc–Smt3 monomers nor His6–Ubc9 (data not shown); on the other hand, the E1-activating enzymes, a hetero- dimeric complex of His6–Aos1 (B40 kDa) and His6–Uba2 (B70 kDa), were still present in the puriﬁed Smt3 chains products. We applied mass spectroscopy to conﬁrm that the band around 30–40 kDa (Figure 1B, lane 4) was His6–Aos1 and not the His6– Myc–Smt3 dimer (data not shown). Smt3 chain-binding assay Equivalent molar amounts of the GST–Red1–His6 (20 mg), GST– Red1I758R–His6 (20 mg) and GST–His6 (4.5 mg) proteins were ﬁrst incubated with 5 ml MagneGST glutathione resin (Promega) in 300 ml binding buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl and 10 mM b-mercaptoethanol) for 3 h. After binding, the resins were washed with phosphate-buffered saline and were suspended again in another 300 ml of binding buffer containing BSA (10 mg) and partially-puriﬁed Smt3 chains (10 mg). The BSA was used to block non-speciﬁc interactions between the Smt3 chains and the glutathione resin. The resin was extensively washed three times with 600 ml wash buffer (50 mM Tris–HCl pH 8.0, 300 mM NaCl and 10 mM b-mercaptoethanol). Protein samples were then separated by SDS–PAGE and analysed by western blotting using an anti-Myc antibody. Supplementary data Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org). Acknowledgements This work was supported by grants to TFW from the National Science Council and Academia Sinica, Taiwan. We thank Michael Lichten (NIH) for the yeast strains used in the physical analyses, Franz Klein (University of Vienna) and Nancy Hollingsworth (Stony Brook University) for the anti-Hop1 antibodies, Scott Keeny (Sloan- Kettering Institute) and Eric Alani (Cornell University) for providing the spo11-HA and spo11da-HA strains, and Franz Klein for personal communication regarding the Hop1 immunostaining results. We give special thanks to Shirleen Roeder and Beth Rockmill (Yale University) for teaching us the meiotic nuclear spreading techni- ques and for providing the BR mutant strains (red1D and hop1D). Conﬂict of interest The authors declare that they have no conﬂict of interest. Bailis JM, Roeder GS (1998) Synaptonemal complex morphogenesis and sister-chromatid cohesion require Mek1-dependent phos- phorylation of a meiotic chromosomal protein. Genes Dev 12: 3551–3563 Bailis JM, Roeder GS (2000) Pachytene exit controlled by reversal of Mek1-dependent phosphorylation. Cell 101: 211–221 594 The EMBO Journal VOL 29 | NO 3 | 2010 & 2010 European Molecular Biology Organization