Red1 binds Smt3 chains F-M Lin et al Figure 3 The Red1–Smt3 chain interaction promotes interhomologue (IH)-recombination product formation. (A) Southern blotting of DNA isolated from meiotic cultures. The diploid cells contained 3.5-kb URA3-ARG4 inserts in LEU2 on one chromosome III homologue (P1) and HIS4 on the other (P2). EcoRI–PvuII double digests probed with HIS4 sequences reveal the crossover (CO1) and non-CO (NCO) products of arg4- EcPal (Clyne et al, 2003). (B, C) CO1 and NCO signal/total lane signal from the Southern blotting in (A). 86% of V5-RED1)4red1D (3.8%; 40% of V5-RED1) (Supplementary Figure S5). We also investigated the overall distribution and amount of DSBs along chromosome VII. DSBs along chromosome VII were detected by Southern blotting using a CRM1 (YGR218W) DNA probe. CRM1 is located near the end of the right arm of chromosome VII (Figure 4A). We found that all three red1 mutants produced fewer DNA DSBs along the entire length of chromosome VII compared with the V5-RED1 cells (Figure 4B and C). The relative order for the overall quantity of DSBs along the chromosome in these strains was V5-RED1 (74.2%)4V5- red1I758R (42.3%; 57% of V5-RED1)BV5-red13R (48.2%; 65% of V5-RED1)4red1D (24.0%; 32% of V5-RED1) (Figure 4D). Although the V5-red1I758R and V5-red13R mutants form fewer DSBs than the wild type cells, it is incorrect to infer that an overall reduction in the DSB number is the only cause of their meiotic phenotypes. It was reported that an allelic series of spo11 mutants (spo11-HA, spo11yf-HA, spo11da-HA) formed B80, B30 and B20% of DSBs along chromosome III compared with wild-type cells, respectively (Martini et al, 2006). Owing to CO homoeostasis, reducing DSBs does not reduce Cos; the CO numbers are maintained at the expense of NCOs. As a result, spo11-HA, spo11yf-HA and spo11da-HA mutants produce 94, 75 and 70% viable spores, respectively (Martini et al, 2006). We conﬁrmed that Hop1 phosphoryla- tion occurs in the spo11-HA and spo11da-HA mutants. The relative order of Hop1 phosphorylation in these strains was SPO11Bspo11-HA4spo11da-HA4spo11D (Figure 4E). As described above, no phosphorylated Hop1 was detected in the V5-red1I758R and V5-red13R mutants (Figure 2A and B). 590 The EMBO Journal VOL 29 | NO 3 | 2010 Because the two SIM mutants generated B60% of the DSB levels of the wild type, it was not surprising that they yielded fewer NCOs (Figure 3C). Owing to CO homoeostasis, these two SIM mutants should produce similar levels of COs as the wild-type V5-RED1 cells. However, these two SIM mutants produced much fewer COs (Figure 3B) and no viable spores. We conclude that a complete loss of Hop1 phosphorylation in these two SIM mutants is likely the main cause for their defects in CO production and spore viability. Detection of Smt3 chains during meiotic mid-prophase Like other known SUMO E3 ligases, Zip3 can promote Ubc9- catalyzed Smt3 polymerisation (Cheng et al, 2006). To examine if the wild-type cells produce Smt3 chains in vivo at the time of SC formation, we constructed a strain that expresses both V5-Smt3 and His6–Myc–Smt3 and a strain that expresses both V5-Smt3-allR and His6–Myc–Smt3. Total cell lysates (TCLs) were collected by trichloroacetic acid (TCA) precipitation during mid-prophase. The lysates were solubi- lized in a denaturing buffer containing 8 M urea and puriﬁed with Ni2 þ -resin, which selectively retains the His6-tagged polypeptides. The TCLs and the Ni2 þ -resin eluants (NREs) were analysed by western blotting. Diploid cells expressing only the V5-Smt3 proteins were used as a negative control for Ni2 þ -resin puriﬁcation. The NREs of the V5-SMT3/HIS6– MYC–SMT3 diploid cells contained both V5-Smt3 and His6–Myc–Smt3. In contrast, far fewer V5-Smt3-allR proteins were co-puriﬁed with His6–Myc–Smt3 using Ni2 þ -resin (Figure 5). These results indicate that Smt3 chains exist at the time of SC formation. The majority of these Smt3 & 2010 European Molecular Biology Organization