Yeast axial-element protein, Red1, binds SUMO chains to promote meiotic interhomologue recombination and chromosome synapsis
2009; Springer Nature; Volume: 29; Issue: 3 Linguagem: Inglês
10.1038/emboj.2009.362
ISSN1460-2075
AutoresFeng-Ming Lin, Yi-Ju Lai, Hui-Ju Shen, Yun-Hsin Cheng, Ting‐Fang Wang,
Tópico(s)Fungal and yeast genetics research
ResumoArticle3 December 2009Open Access Yeast axial-element protein, Red1, binds SUMO chains to promote meiotic interhomologue recombination and chromosome synapsis Feng-Ming Lin Feng-Ming Lin Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Yi-Ju Lai Yi-Ju Lai Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Hui-Ju Shen Hui-Ju Shen Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Yun-Hsin Cheng Yun-Hsin Cheng Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Ting-Fang Wang Corresponding Author Ting-Fang Wang Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Feng-Ming Lin Feng-Ming Lin Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Yi-Ju Lai Yi-Ju Lai Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Hui-Ju Shen Hui-Ju Shen Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Yun-Hsin Cheng Yun-Hsin Cheng Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Ting-Fang Wang Corresponding Author Ting-Fang Wang Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Author Information Feng-Ming Lin1,2, Yi-Ju Lai1,2, Hui-Ju Shen2, Yun-Hsin Cheng2 and Ting-Fang Wang 1,2 1Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan 2Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan *Corresponding author. Institute of Molecular Biology, Academia Sinica, 128 Academia Road, Nankang, Taipei 11529, Taiwan. Tel.: +886 2 2789 9188; Fax: +886 2 2782 6085; E-mail: [email protected] The EMBO Journal (2010)29:586-596https://doi.org/10.1038/emboj.2009.362 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The synaptonemal complex (SC) is a tripartite protein structure consisting of two parallel axial elements (AEs) and a central region. During meiosis, the SC connects paired homologous chromosomes, promoting interhomologue (IH) recombination. Here, we report that, like the CE component Zip1, Saccharomyces cerevisiae axial-element structural protein, Red1, can bind small ubiquitin-like modifier (SUMO) polymeric chains. The Red1–SUMO chain interaction is dispensable for the initiation of meiotic DNA recombination, but it is essential for Tel1- and Mec1-dependent Hop1 phosphorylation, which ensures IH recombination by preventing the inter-sister chromatid DNA repair pathway. Our results also indicate that Red1 and Zip1 may directly sandwich the SUMO chains to mediate SC assembly. We suggest that Red1 and SUMO chains function together to couple homologous recombination and Mec1–Tel1 kinase activation with chromosome synapsis during yeast meiosis. Introduction Crossing-over between homologous chromosomes ensures accurate segregation during the first meiotic division. Homologous chromosomes must first be linked by chiasmata, the cytological manifestation of crossover (CO) recombination products that are established during meiotic prophase. Meiotic recombination is initiated by developmentally programmed DNA double-strand breaks (DSBs), the formation of which requires the products of at least 10 genes, including SPO11 (Keeney, 2001). These DSBs are then resected by the Mre11-Rad50-Xrs2 nuclease to generate 3′ single-strand tails that invade the intact DNA duplexes used in DNA repair (Neale et al, 2005). Most of these events use homologous chromosomes, not sister chromatids, as the templates in DNA repair, yielding CO and non-CO (NCO) products (for a review see Bishop and Zickler, 2004). Two kinds of CO are seen in Saccharomyces cerevisiae. The first CO class is randomly distributed along the chromosomes and requires Mus81-Mms4 endonuclease (de los Santos et al, 2003; Borner et al, 2004; Lynn et al, 2007). The second CO class is distance-dependent and requires ZMM proteins (an acronym for yeast proteins Zip1-4, Msh4-5 and Mer3; Borner et al, 2004) at CO-designated sites to stabilise early recombination intermediates against the action of the anti-CO helicase Sgs1 (Jessop et al, 2006; Oh et al, 2007, 2008; Jessop and Lichten, 2008). The ZMM proteins also form a zipper-like proteinaceous structure between homologous chromosomes, known as the synaptonemal complex (SC), during the zygotene and pachytene stages. These structures include a major SC component (Zip1) and the SC initiating or elongating proteins (Zip2-4, Mer3, Msh4-5 and Spo16) (Sym et al, 1993; Hollingsworth et al, 1995; Chua and Roeder, 1998; Agarwal and Roeder, 2000; Novak et al, 2001; Borner et al, 2004; Fung et al, 2004; Perry et al, 2005; Tsubouchi et al, 2006; Shinohara et al, 2008). The SC consists of a central region and two dense lateral elements. The lateral element constitutes the rod-like homologue axis, which is called an axial element (AE) before synapsis. The chromatin loops of sister chromatids are organised along the axis. The central region contains transverse filaments (TFs) oriented perpendicularly to the longitudinal axis of the SC, resulting in the striated, zipper-like appearance of the SC (Henderson and Keeney, 2005). The components of the yeast AE include sister chromatid cohesin complexes (e.g., Rec8), DNA topoisomerase II (Top2) and a few meiosis-specific proteins (e.g., Red1, Hop1 and Mek1) (Hollingsworth and Ponte, 1997; Smith and Roeder, 1997; Klein et al, 1999; Blat et al, 2002; Page and Hawley, 2004). Mek1 is a serine–threonine protein kinase that functions in combination with Red1 and Hop1 to ensure interhomologue (IH) recombination by preventing the use of a sister chromatid as the template in DNA repair (Bailis and Roeder, 1998; Wan et al, 2004; Niu et al, 2005, 2007). Mek1 kinase activity is dependant on Hop1 phosphorylation, which is mediated by Mec1 and Tel1, yeast homologues of the mammalian ATR and ATM kinases (Carballo et al, 2008). Hop1 protein has been shown to interact with DNA in vitro (Kironmai et al, 1998) and to interact with itself and Red1 (Bailis and Roeder, 1998; de los Santos and Hollingsworth, 1999; Woltering et al, 2000). Hop1 also promotes dimerisation of Mek1 (Niu et al, 2005). Red1 is known to interact with itself, Hop1 and the protein phosphatase, Glc7 (Bailis and Roeder, 2000; Woltering et al, 2000). Zip1 is the major structural component of the central region (Sym et al, 1993), The Zip1 proteins form head-to-head dimers connected by protein–protein interactions between the central coiled-coil domains, and the C-terminal globular domain connects the central region to the AEs (Dong and Roeder, 2000). The SC initiation protein Zip3 is an E3 ligase for Smt3, the yeast small ubiquitin-like modifier (SUMO). Post-translational modifications of SUMO control SC assembly through an interaction between the Zip1 C-terminal globular domain (residues 849–875) and the Zip3-dependent Smt3 conjugates along two AEs (Cheng et al, 2006). In the wild-type cell, Zip3 recruits Ubc9, the SUMO E2 enzyme, to meiotic chromosomes, but in the zip3Δ mutant, Ubc9 proteins are not associated with chromosomes (Hooker and Roeder, 2006). However, the Ubc9 proteins in mutant cells still catalyse the self-polymerization of Smt3 monomers to form Smt3 chains (Cheng et al, 2006). These chromosome-free Smt3 chains non-covalently associate with Zip1 proteins to form one or a few large proteinaceous aggregates known as polycomplexes (PCs). Zip1 stabilizes these chromosome-free Smt3 chains against the Ulp2 SUMO protease, resulting in a massive accumulation of Smt3 chains (Cheng et al, 2006). Other ZMM proteins (i.e., Zip2, Spo16) have been proposed to act after Zip3 as chaperone-like machinery to facilitate the perpendicular alignment of Zip1 proteins along two AEs during SC elongation. The meiotic chromosomes of these two zmm mutants can recruit Zip1, but are barely able to form long and fine Zip1 lines between homologous chromosomes (Cheng et al, 2006). This observation is consistent with a recent report that Zip2 functions in SC elongation is dependant upon SPO11 (Tsubouchi and Roeder, 2005; Tsubouchi et al, 2008). Spo11 generates DSBs, subsequently promoting Hop1 phosphorylation to ensure the close juxtaposition of two homologous chromosomes, by IH recombination, for SC elongation (Niu et al, 2005; Carballo et al, 2008). Zip1 also mediates pair-wise non-homologous centromere coupling (NHCC) before the zygotene stage (Tsubouchi and Roeder, 2005). Zip1 binds to centromeric Smt3 conjugates (e.g., Top2), which is mediated by non-Zip3 SUMO E3 ligases, Siz1, Siz2 or Mms21. These non-Zip3 E3 ligases are downregulated after the zygotene stage. The NHCC is then destroyed by SUMO protease Ulp2 (Cheng et al, 2006). NHCC could lead to SC initiation at the centromeres. These centromeric SCs are independent of SPO11 and ZIP3, which have major roles in the chromosome synapsis at non-centromeric locations. On the other hand, Spo11 and Zip2 are required for SC elongation at both centromeric and non-centromeric sites (Tsubouchi and Roeder, 2005; Tsubouchi et al, 2008). These two kinds of SCs are initiated differently but likely use the same mechanism for SC elongation. As described above, Spo11 is required for Hop1 phosphorylation, which ensures IH-recombination by repressing IS-recombination (Niu et al, 2005; Carballo et al, 2008). However, DSB-dependent Hop1 phosphorylation is not a prerequisite for SC initiation, because Zip1 and Zip3 can be recruited to meiotic chromosomes in the absence of SPO11, for example, a previous study found that 67% of Zip3 foci were centromeric and 33% were non-centromeric (Tsubouchi et al, 2008). The function of DSB-dependent Hop1 phosphorylation at non-centromeric locations is to closely juxtapose two homologous chromosomes for SC initiation and elongation, whereas the function of DSB-independent NHCC at centromeres is to initiate SC assembly between two non-homologous chromosomes (Tsubouchi et al, 2008). Therefore, we reason that other non-Hop1 AE structural proteins may be responsible for Zip3- and Smt3-dependent SC initiation. In this study we found that, like Zip1, Red1 can bind Smt3 chains. The Red1–Smt3 interaction first facilitated chromosome recruitment of Zip1 and Zip3 for SC initiation, similar to the function of centromeric Smt3 conjugates in recruiting Zip1 and Zip3 proteins during NHCC (Tsubouchi and Roeder, 2005; Cheng et al, 2006; Tsubouchi et al, 2008). The second function of the Red1–Smt3 interaction was to promote Hop1 phosphorylation, which prevents IS-recombination to ensure IH-recombination. As a result, the two homologous chromosomes are closely juxtaposed for Zip2 or other ZMM proteins to facilitate the perpendicular alignment of Zip1 proteins during SC elongation (Tsubouchi and Roeder, 2005; Cheng et al, 2006; Tsubouchi et al, 2008). Results Red1 binds Smt3 chains but not to Smt3 monomer A previous genome-wide two-hybrid screen suggested that Red1 might interact with Smt3 (Ito et al, 2001). However, it was unclear if the Red1–Smt3 two-hybrid interaction was because of a direct physical interaction between Red1 and Smt3 monomer, Smt3 polymeric chain or conjugate. Moreover, this two-hybrid interaction may have indirectly resulted from post-translational Smt3 modification of Red1 itself or some other Red1 binding protein. The latter hypothesis is not favoured for the following reasons: first, it is inconsistent with the result that the zip3Δ mutant could form short SCs in the absence of the Zip3-dependent Smt3 modified AE proteins (Agarwal and Roeder, 2000; Hooker and Roeder, 2006); second, very little Red1 protein ( Red1C2R>Red1CI758R>Red1C3R (Figure 1A), indicating that SIM-2 is better than SIM-1 at binding Smt3. Red1C did not exhibit a two-hybrid interaction with Smt3-ΔGG, a conjugation-incompetent Smt3 mutant lacking the C-terminal pair of glycines for E1-mediated Smt3 activation (for a review see Gill, 2004). In addition, Red1C and Smt3 induced a high level of two-hybrid interaction in smt3-allR reporter cells. However, Red1C and Smt3-allR exhibited a weaker interaction in the same reporter cells (Figure 1A). Smt3-allR is competent in the SUMO conjugation to all target proteins, including the wild-type Smt3. However, Smt3-allR cannot form a polymeric chain because the nine lysine residues in Smt3 were replaced with arginines in this mutant (Bylebyl et al, 2003). These results suggest that Red1 preferentially associates with Smt3 chains but not with Smt3 monomer or other Smt3 conjugates. Figure 1.Red1 preferentially binds Smt3 chains. (A) Quantitative two-hybrid assays were carried out using mitotic reporter host cells carrying either SMT3 or mutant smt3-allR gene. One unit of β-galactosidase hydrolyses 1 μmol of o-nitrophenyl β-D-galactopyranoside per min per OD600 unit. Red1C represents the C-terminal fragment (residues 611–827) of the wild-type Red1 protein. The Ile758 residue of SIM-2 was converted to Arg in the Red1CI758R mutant; Red1C2R has two point mutations (Ser715 to Arg and Thr717 to Arg) at the SIM-1. The Red1C3R is the synthetic mutant of the Red1CI758R and Red1C2R mutants. (B) Purified GST–His6, GST–Red1–His6, GST–Red1I758R–His6, and His6–Myc–Smt3 chains were separated by SDS–PAGE and visualised by Coomassie blue staining. (C) Purified GST–His6, GST–Red1–His6 and GST–Red1I758R–His6 fusion proteins were first bound to GST resins and then incubated with purified His6–Myc–Smt3 polymeric chains. After extensive washing, the proteins bound to the GST resins were visualised by western blot using anti-Myc antibody. Download figure Download PowerPoint To examine if the Red1–Smt3 chain interaction is essential for meiosis, we constructed five yeast strains expressing V5-tagged Red1 (V5-Red1), V5-Red12R, V5-Red1I758R, V5-Red13R or no Red1 protein. Tetrad analyses revealed that the V5-RED12R mutant, like V5-RED1, generated many viable spores (>97%), whereas the V5-red1I758R, V5-red13R and red1Δ mutants yielded almost no viable spores (<1%). We conclude that SIM-2 not only exhibits a stronger interaction with Smt3 chains than SIM-1, but it is also functionally more important in vivo. Finally, we examined the affinity of Red1 variant proteins for Smt3 chains. We expressed full-length Red1 fusion proteins GST–Red1–His6 and GST–Red1I758R–His6 in Escherichia coli, and then sequentially purified them on Ni2+ and glutathione resins. Smt3 chains were synthesised in vitro and partially purified by size-exclusion gel filtration (Figure 1B). The order of affinity of these proteins had for Smt3 chains was GST–Red1–His6≫GST–Red1I758R–His6 ∼ GST–His6 (Figure 1C). Red1CI758R and Red1C3R were also partly defective in homo-oligomerization in the two-hybrid assays carried out in SMT3 and smt3-allR reporter cells, respectively (Supplementary Table S1). Although the results obtained from the smt3-allR reporter cells indicate that the Red1–Red1 interaction is not likely mediated by Smt3 chains, it does not exclude the possibility that other proteins conjugated by Smt3-allR or Smt3 may facilitate the Red1–Red1 interaction. Nevertheless, these two-hybrid results still raise a concern that Red1 homo-oligomerization (Woltering et al, 2000) may be required for the normal functioning of Red1 protein, including the Red1–Smt3 chain interaction. This supposition is not favourable, because the GST–Red1–His6 proteins exhibited much higher affinity to Smt3 chains than the GST–Red1I758R–His6 proteins exhibited (Figure 1C). GST is a dimeric protein on its own, it was used here to induce homo-oligomerization of GST–Red1–His6 and GST–Red1I758R–His6 recombinant proteins. Red1 binds Smt3 chains to promote meiotic IH-recombination The V5-red1I758R and V5-red13R mutant cells had lower steady-state levels of Red1 protein (roughly 70%) compared with wild-type cells (Figure 2A). To ensure that these two mutants did not produce less Red1 proteins than the wild-type V5-RED1 cells, high-copy number vectors expressing V5-Red1I758R or V5-Red13R were then transformed into V5-red1I758R or V5-red13R mutants, respectively (Figure 2B). All of these mutant strains exhibited identical meiotic phenotypes (i.e., <1% spore viability) before and after being transformed with the supplementary V5-Red1I758R and V5-Red13R expression vectors. Therefore, the meiotic defects of the V5-red1I758R and V5-red13R cells were not because of lower steady-state levels of Red1 proteins. Figure 2.Characterisation of the V5-red1I758R and V5-red13R proteins. (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, respectively. 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 phenotypes 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 μg/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. Download figure Download PowerPoint We then carried out cycloheximide shut-off experiments (Penkner et al, 2005) to examine Red1 protein stability. Protein synthesis was inhibited by 200 μg/ml of cycloheximide added into 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. Western blot analysis revealed that the levels of V5-Red1, V5-Red1I758R and V5-Red13R remained steady in the presence of cycloheximide upto 180 min (Figure 2D), indicating that the SIM mutation(s) has little or no effect on V5-Red1 protein stability. Immunostaining of sporulating cells and meiotic nuclear spreads with an anti-V5 antibody revealed that V5-Red1I758R and V5-Red13R mutant proteins were properly targeted to nuclei (Supplementary Figure S1) and meiotic chromosomes (Supplementary Figure S2), respectively. We also confirmed that these two mutants were not defective in Hop1 chromosomal localisation (Supplementary Figure S2) or the Red1–Hop1 interaction (Woltering et al, 2000) (Supplementary Table S2 and Figure S3). Our results also revealed that Red1 proteins in the V5-red1I758R and V5-red13R mutant cells were hypophosphorylated 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 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 phosphorylation-deficient mutants undergo normal meiosis and generate 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 red1Δ null 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 red1Δ 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 chromatid 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 red1Δ mutant, yielded no viable spores ( V5-red1I758R (3.7; 30% of V5-RED1)∼V5-red13R (4.4%; 35% of V5-RED1)∼red1D (2.1%; 17% of V5-RED1) (Figure 3B), whereas the relative amount of total NCO products was V5-RED1 (7.3%)>V5-red1I758R (3.9%; 53% of V5-RED1)∼V5-red13R (4.1%; 57% of V5-RED1)>red1D (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). 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). Download figure Download PowerPoint 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 red1Δ mutants. Genomic DNA isolated from sporulating cultures at various time points was digested with BglII, separated by gel electrophoresis, 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%)∼V5-red1I758R (9.2%; 97% of V5-RED1)∼V5-red13R (8.2%; 86% of V5-RED1)>red1Δ (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%)>V5-red1I758R (42.3%; 57% of V5-RED1)∼V5-red13R (48.2%; 65% of V5-RED1)>red1Δ (24.0%; 32% of V5-RED1) (Figure 4D). 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 (∼1100 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 spo11Δ cells at the 7-h meiotic time point. Download figure Download PowerPoint 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 ∼80, ∼30 and ∼20% 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 phosphorylation occurs in the spo11-HA and spo11da-HA mutants. The relative order of Hop1 phosphorylation in these strains was SPO11∼spo11-HA>spo11da-HA>spo11Δ (Figure 4E). As described above, no phosphorylated Hop1 was detected in the V5-red1I758R and V5-red13R mutants (Figure 2A and B). Because the two SIM mutants generated ∼60% 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 solubilized in a denaturing buffer containing 8 M urea and purified with Ni2+-resin, which selectively retains the His6-tagged polypeptides. The TCLs and the Ni2+-resin eluants (NREs) were analysed by western blotting. Diploid cells expressing only the V5-Smt3 proteins were used as a negative control for Ni2+-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+-resin (Figure 5). These results indicate that Smt3 chains exist at the
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