Figure 2 Characterisation of the V5-red1I758R and V5-red13R pro- teins. (A) Western blot time-course analysis of the sporulation cells using anti-V5 antibody. The lower and upper bands represented the non-phosphorylated and phosphorylated V5-Red1 proteins, respec- tively. The blot was also probed with anti-Hop1 antibody to determine if Hop1 protein is phosphorylated. The results indicate that the steady-state levels of Red1 proteins in the V5-red1I758R and V5-red13R cells were lower than the level in V5-RED1 cells. Arp7 was used as a loading control. The molecular weights (in kDa) are indicated to the left of the blots. (B) To exclude the possibility that the meiotic defects of these two SIM mutants resulted from a lower steady-state level of mutant Red1 proteins, we constructed two yeast expression vectors to over-express V5-Red1I758R and V5-red13R proteins under control of the GAL1 promoter. Each vector was transformed into the corresponding yeast strain, V5-red1I758R or V5-red13R cells, respectively. We added 0.03% galactose into the sporulation medium to induce the overexpression of V5-Red1I758R or V5-red13R protein. We conﬁrmed that the presence of 0.03% galactose did not affect either the sporulation efﬁciency or spore viability of the V5-RED1 cells. These two new mutant yeast strains each produced a similar or higher steady-state level of Red1 proteins compared to the wild-type V5-RED1 cells. Tetrad analysis revealed that these two new yeast strains, like the original strains, could not form viable spores. These results indicate that the meiotic pheno- types of the original V5-red1I758R and V5-red13R cells were not likely owing to a decreased amount of Red1 protein. (C) Cycloheximide shut-off experiment to demonstrate that V5-Red1I758R and V5-red13R proteins are as stable as the V5-Red1 proteins. Protein synthesis was inhibited by 200 mg/ml cycloheximide added to the meiotic cultures at the 4-h time point. Samples were taken at 0, 30, 60, 90, 120 and 180 min after the addition of cycloheximide. Our results also revealed that Red1 proteins in the V5-red1I758R and V5-red13R mutant cells were hypopho- sphorylated compared with wild-type cells (Figure 2A and B) (Lai YJ et al, unpublished data), raising a concern that the defects in these SIM mutants may be because of a deﬁciency in protein phosphorylation. Although phosphorylated Red1 proteins were thought to have a critical role in meiosis (Bailis & 2010 European Molecular Biology Organization Red1 binds Smt3 chains F-M Lin et al and Roeder, 1998; de los Santos and Hollingsworth, 1999; Hong and Roeder, 2002; Wan et al, 2004), we have mapped the Red1 phosphorylation sites and showed that phosphor- ylation-deﬁcient mutants undergo normal meiosis and gen- erate as many viable spores as wild-type V5-RED1 cells (Lai YL et al, unpublished data). In summary, we discovered a new essential biochemical property of Red1 protein. The defects in the two SIM mutant proteins, V5-red1I758R and V5-red13R, are mainly due to the loss of the Red1–Smt3 chain interaction rather than a global loss of the function of the C-terminal region of the proteins (including protein stability, protein phosphorylation, Red1– Red1 oligomerization and Red1–Hop1 interaction or nuclear/ chromosomal targeting). Accordingly, these two SIM mutant strains are not identical to the red1D mutant. Red1 binds Smt3 chains to promote Hop1 phosphorylation Western blot time-course analyses also showed a signiﬁcant portion of Hop1 proteins are phosphorylated in sporulating V5-RED1 cells. Phosphorylated Hop1 migrated more slowly in the SDS–PAGE than unmodiﬁed Hop1. In contrast, Hop1 was not phosphorylated in the V5-red1I758R, V5-red13R or red1D cells (Figure 2A and B). These results indicate that Red1 binds Smt3 chains to promote Tel1- and Mec1-dependent Hop1 phosphorylation. Hop1 phosphorylation is known to be essential for the activation of Mek1 kinase activity and ensures IH recombination by preventing the inter-sister chro- matid DNA repair pathway (Niu et al, 2005, 2007; Carballo et al, 2008). These results are consistent with our ﬁnding that V5-red1I758R and V5-red13R, like the red1D mutant, yielded no viable spores (o1%). A physical assay was carried out to monitor IH-recombina- tion products (i.e., COs and NCOs) (Clyne et al, 2003). Yeast genomic DNA from different meiotic time points was digested with the restriction enzymes EcoRI and PvuII and analysed by Southern blotting. The V5-RED1 diploid cell produced both CO and NCO products, though only B50% as much NCOs as COs (Figure 3A). The relative amount of total CO products produced by these four strains was V5-RED1 (12.4%)4 V5-red1I758R (3.7; 30% of V5-RED1)BV5-red13R (4.4%; 35% of V5-RED1)Bred1D (2.1%; 17% of V5-RED1) (Figure 3B), whereas the relative amount of total NCO products was V5-RED1 (7.3%)4V5-red1I758R (3.9%; 53% of V5-RED1)B V5-red13R (4.1%; 57% of V5-RED1)4red1D (1.5%; 21% of V5-RED1) (Figure 3C). We also conﬁrmed that overexpression of the SIM mutant proteins in relevant strains did not affect the formation of COs and NCOs (Supplementary Figure S4). To determine if the loss of the Red1–Smt3 chain interaction might also affect the amount of DSBs generated during meiosis, we ﬁrst examined DSB formation at YCR047C, a DSB hot spot on chromosome III. To quantify the level of DSBs more precisely, we introduced the rad50S mutation, which blocks the resection of the DSB ends, into the V5-RED1, V5-red1I758R, V5-red13R and red1D mutants. Genomic DNA isolated from sporulating cultures at various time points was digested with BglII, separated by gel electro- phoresis, blotted and hybridised with the YCR052W DNA probe (Buhler et al, 2007). The order for the overall quantity of DSBs at this speciﬁc site was V5-RED1 (9.5%)B V5-red1I758R (9.2%; 97% of V5-RED1)BV5-red13R (8.2%; The EMBO Journal VOL 29 | NO 3 | 2010 589