Red1 binds Smt3 chains F-M Lin et al Figure 4 The overall distribution of double-strand breaks (DSBs) along chromosome VII on a rad50S background. DSBs along chromosome VII were detected by Southern blotting analysis using a CRM1 (YGR218W) DNA probe. CRM1 is located near the end of the right arm of chromosome VII. (A) Schematic drawing of chromosome VII (B1100 kb). The CRM1 and CEN7 loci are indicated. (B) Yeast chromosomes were separated by pulse-ﬁeld gel electrophoresis, analysed by Southern blotting with a CRM1 DNA probe, and visualised using a phosphoimager. The three red1 mutants produced equal or fewer DNA DSBs along the entire length of chromosome VII compared with the V5-RED1 cells. (C) Blots were quantiﬁed using a Fujiﬁlm image analysis program (Image Gauge 4.0). The plots show traces of representative lanes at 0 h (upper panel), 9 h (middle panel), and 12 h (lower panel), respectively. (D) DSBs are expressed as a percent of the total radioactivity in the lane after background subtraction, not including material in the wells. (E) Western blot detection of Hop1 and Spo11-HA proteins in the SPO11, spo11-HA, spo11-da-HA, and spo11D cells at the 7-h meiotic time point. chains have apparent molecular weights of approximately 100 000–140 000 Da on western blot analysis (Figure 5, lanes e and k). Because the apparent molecular weights of V5-Smt3 and the His6–Myc–Smt3 monomers are roughly 20 000– 24 000 Da, these Smt3 chains likely consist of ﬁve or six Smt3 monomers. Alternatively, the proteins may be shorter Smt3 chains conjugated on other novel non-Smt3 protein(s). Red1 and Zip1 sandwich Smt3 chains to mediate SC assembly Zip1 and Zip3 each contain a SIM for direct interaction with Smt3 conjugates (Cheng et al, 2006). This observation is consistent with reports that the chromosomal recruitment of Zip1 and Zip3 for SC initiation occurs in the absence of SPO11 (Tsubouchi and Roeder, 2005; Tsubouchi et al, 2008). Spo11 functions (i.e., DSB formation) is a prerequisite for & 2010 European Molecular Biology Organization Hop1 phosphorylation, which ensures IH-recombination through repression of IS-recombination (Niu et al, 2005; Carballo et al, 2008). Therefore, a lack of Hop1 phosphoryla- tion or failure to prevent IS-recombination may not fully account for the SC assembly defects in meiotic V5-red1I758R and V5-red13R cells. We previously reported that Zip1 and Red1 interacted in a two-hybrid assay (Cheng et al, 2006). Unfortunately, we were unable to co-immunoprecipitate Zip1 with V5-Red1 from meiotic cell lystaes (data not shown), because the Zip1 proteins, unlike Red1 or Hop1, became extremely unstable after the meiotic cells were lysed in native buffers containing a variety of protease inhibitors (Supplementary Figure S6). More than 30 years ago, vacuolar proteases were reported to activate upon cell lysis in native buffers and the SDS sample buffer, leading to widespread proteolysis of yeast The EMBO Journal VOL 29 | NO 3 | 2010 591