Figure 1.
Smc6, but not Mms21 SUMO ligase activity is required for chromosome segregation after meiotic recombination.
(A) Schematic representation of the Smc5/6-Mms21 subunits. (B) Schematic of the temperature shift experiments. To avoid mitosis and premeiotic S-phase a temperature shift from 23.5 to 33°C was carried out in increments upon induction of meiosis (see experimental procedures) reaching 33°C by 2.5 hours in SPM when cells are past bulk DNA replication. This scheme eliminates Smc6 at the point when the earliest DSBs become detectable and interference with mitosis and pre-meiotic S-phase is little. (C) DNA staining with DAPI after spore formation. The right micrograph shows the presence of 4 nuclei and mitochondrial DNA in a wild type tetrad. The left micrograph shows the nuclear DNA outside the four spores, pressed against the ascus wall, while only the mitochondrial DNA had segregated into the spores. Left table: Percent of asci with 0,1,2,3,4 spores containing nuclear DNA (n = 200 tetrads). (D) Spindle staining by anti-tubulin. The upper panel shows examples of spindle morphologies post anaphase I in wild type. The lower panel shows aberrant spindles of the smc6-56 mutant, consistent with physical impediment of DNA separation. (E) Meiotic nuclear divisions are blocked in smc6-56 mutants at 33°C. Left panel: wild type (33°C), middle: mms21-11 (30°C), right: smc6-56 (33°C). Empty black circles: cells containing one DAPI stained nucleus (1n), filled red circles: cells with 2 nuclei (2n), filled green circles: cells with 4 nuclei (4n). (F) Meiotic progression is normal in smc6-56 (33°C): Meiotic progression was followed by spindle morphology by tubulin labeling in an in situ staining procedure. Monopolar and bi-polar spindles were plotted separately against hours in SPM. n = 200 cells per experiment, continuous lines: monopolar spindles, dotted lines: bipolar spindles. Green: Wild type (30°C), black: mms21-11 (30°C), red: smc6-56 (33°C), blue: mnd1Δ smc6-56 (33°C). (G) spo11Δ suppresses the chromatin separation defect of smc6-56. Cells containing at least 2 separated nuclei plotted against the time in sporulation. Grey filled circles: smc6-56 SPO11, blue filled circles: smc6-56 spo11Δ, red filled circles: smc6-56 spo11Δ spo13Δ.
Figure 2.
The Smc5/6-Mms21 complex binds early to meiotic chromatin and associates with sites of DSB repair.
(A) Co-immuno labeling for Smc6-myc13 and cohesin subunit Rec8-HA3 of a spread, leptotene nucleus and pachytene nucleus. Red: Smc6-myc13, Green: Rec8-HA, white bar: 5 µm. (B) Co-immuno labeling for Smc6-myc13 and recombinase Rad51 of a spread meiotic prophase I nucleus. Red: Smc6-myc13, Green: Rad51, white bar: 5 µm. White rectangle indicates position of magnified sub region. (C) Co-immuno labeling for Smc6-myc13 and synapsis specific Zip1 protein of several meiotic stages. (Figure S3 displays a complete series of double stained nuclei, staged according to Zip1 and DNA morphology): Red: Smc6-myc13, Green: Zip1, White (and blue) figures below the immunostained nuclei represent the corresponding nuclear DNA stained with DAPI. White bars: 5 µm. Stages correspond to (from left to right): Early zygotene, late zygotene, pachytene, diplotene, late anaphase I. (D) Smc6-myc13 (t = 3.5 hours in SPM) localizes at DSB sites. ChIP-seq signals on a 130 kb region of chromosome V are shown after smoothing (bandwidth: 500 bp), NCIS normalization and background subtraction as described in Materials and Methods. Red: Smc6-myc13, black: Smc6-myc13, spo11Δ, filled grey profile: Mer2-HAint. (t = 4h) to illustrate core site signals. DSB hotspots defined in [53] were plotted in green on the negative scale to indicate their positions and relative intensities. The diagram on the right shows the results of qPCR at three positions on chromosome III from the same experiment: a DSB site (ca. at 211k, YCR047C), a core site (ca. at 219k) and a cold spot (ca. at 136, ADP1). To correct for possible differences in the efficiencies of the IPs across the different strains, the enrichment of core and DSB qPCR signals relative to the ADP1 signal per ChIP is plotted for the indicated genotypes and time points. Core/ADP1 shown in blue, DSB/ADP1 in red. (E) Same as (D) but Smc6-myc13 analyzed at t = 4.5 hours in SPM. (F) qPCR on chromosome III from the same experiment as in (D,E), a DSB site (ca. at 211k, YCR047C), a core site (ca. at 219k) and a cold spot (ca. at 136, ADP1). To correct for possible differences in the efficiencies of the IPs across the different strains, the enrichment of core and DSB qPCR signals relative to the ADP1 signal is plotted for the indicated genotypes and time points, Core/ADP1 shown in blue, DSB/ADP1 in red. (G) Representation of the rDNA locus on chromosome XII as provided by the Saccharomyces Genome Database (SGD) showing two (of approximately 200) rDNA repeats. Profiles for Smc6-myc13 at 4.5 hours in SPM are shown (blue: WT, black: spo11Δ; smoothed at bandwidth 250bp, decile normalized and background subtracted (minus untagged)). Sharp Smc6 signals flank the 35S rDNA transcriptional unit independent of Spo11. Positions of replication origins (ARS), nontranscribed spacers (NTS) and RDN37 repeats (blue arrows) are indicated below. (H) Centromeres display a Smc6 signal. As in (G) but without background subtraction for a small region around CEN I (white circle). Green: Smc6-myc13, t = 4.5, black: untagged.
Figure 3.
The Smc5/6-Mms21 complex is required to prevent the accumulation of toxic Joint Molecules.
(A) Schematic representation of the genetic loci, restriction sites and the probe used to demonstrate joint molecule formation (modified from [6]). (B) Joint molecules accumulate and persist in smc6-56. Southern blotting of samples taken from synchronous meiotic time courses at restrictive temperature with DNA extracted and digested with Xmn1 under conditions preserving JMs and using the probe indicated in (A). Lane number indicates hours in SPM. Size markers are provided to the left of the blots while the identity of the labeled species is indicated to the right. The bracket indicates the region where various JMs (2 strand (IH and IS), 3 strand or 4 strand JMs) migrate to. P1, P2: parental fragments. Dashed lines highlight the regions on the blot where DSB signals or JM signals appear. Left Panel: Wild type, right panel: smc6-56. (C) Schematic representation of the genetic loci, restriction sites and the probe used to detect and quantify IS-JMs, COs and NCOs (modified from [6]). (D) As in (B) from the same time course experiment but genomic DNA digested with Xho1 and EcoR1 and probe corresponding to (C). Note: hisU probe detects only one parental fragment (P2). (E) JMs accumulate and persist in smc6-56 at restrictive temperature. Total JM signals were quantified, subtracted from background and plotted as % of total signal as a function of time in SPM. Green: Wild type (33°C), red: smc6-56 (33°C), red dotted line: Inter sister JM from the blot shown in (D). (F) Near normal levels of CO in smc6-56: Representation as in (E) but quantification of CO product. (G) Normal levels of NCO in smc6-56: Representation as in (E) but quantification of NCO product.
Figure 4.
Mms21 SUMO E3 ligase activity antagonizes inappropriate Joint Molecule formation in zip3Δ and in mms4-mn mutants.
(A) mms21-11 restores bivalent formation and axial associations in zip3Δ mutants. Shown are representative nuclei after chromosome spreading and immunolabeling with Hop1 to visualize chromosome axes. Top panel: zip3Δ. Bottom panel: zip3Δ mms21-11 double mutant. Red arrows point at a mms21-11 dependent bivalent with apparently two axial associations. Several such bivalents emerge in this nucleus. (B) Quantification of bivalent formation: Spread nuclei were classified according to the number of bivalents present. The discrete density of the distribution is plotted (incidences against class). n = 100 nuclei were classified per experiment. Red: zip3Δ, green and blue: two biological repeats of zip3Δ mms21-11. (C) mms21-11 improves meiotic progression in zip3Δ: Meiotic progression was followed via spindle morphology. Monopolar and bi-polar spindles were plotted against hours in SPM. n = 200 cells per experiment, continuous lines: monopolar spindles, dotted lines: bipolar spindles, red: zip3Δ, green: zip3Δ mms21-11. (D) mms21-11 improves spore viability in zip3Δ: Spore viability was assayed by tetrad dissection in n = 40 tetrads per mutant, red: zip3Δ, green: zip3Δ mms21-11. (E) JMs accumulate in mms21-11 that require the Mus81-Mms4 resolvase for resolution: Same JM, CO and NCO assay as described in Figure 3 (A through D). Southern blotting of samples from synchronous meiotic time courses at 30°C. Upper panels: Xmn1 digest, JM detection. Left: mms21-11, Middle: mms21-11 mms4-mn, Right: mms4-mn. Lower panels: Xho1/EcoR1 digest, CO/NCO detection. Left: mms21-11, Middle: mms21-11 mms4-mn, Right: mms4-mn. (F) Meiotic progression is normal in all mms21-11 and mms4-mn mutants: Meiotic spindles were labeled. Monopolar spindles were plotted against hours in SPM. n = 200 cells per experiment, continuous lines: monopolar spindles. Red: mms4-mn mms21-11, black: mms21-11, blue: mms4-mn. (G) Meiotic nuclear divisions are blocked in mms4-mn mms21-11 double mutants. Continuous lines: cells containing one DAPI stained nucleus (1n), dotted lines: cells with 2 or 4 nuclei/DAPI-bodies (2n+4n). Red: mms4-mn mms21-11, black: mms21-11, blue: mms4-mn. (H) Segregation of DNA into spores: Numbers represent the percentage of tetrads with either DNA in all 4 spores (upper 3 lines), or all chromosomes outside the spores (lower 3 lines). (I) JMs accumulate and persist in mms4-mn mms21-11 double mutants. Total JM signals were quantified, subtracted from background and plotted as % of total signal as a function of time in SPM. Red: mms4-mn mms21-11, black: mms21-11, blue: mms4-mn, red dashed line: smc6-56 (33°C). (J) Reduced levels of CO in mms4-mn mms21-11 double mutants: Representation as in (I) but quantification of CO product. (K) Reduced levels of NCO in mms4-mn mms21-11 double mutants: Representation as in (I) but quantification of NCO product.
Figure 5.
The Smc5/6-Mms21 complex is required for the function of the “rogue JM resolvases”.
(A) Rogue JMs forming in sgs1-mn require Smc6 for their resolution: Same JM assay as described in Figure 3 (A, B). Southern blotting for JMs from synchronous meiotic time courses at 33°C. Xmn1 digest, JM detection. Left: sgs1-mn, Right: sgs1-mn smc6-56. (B) CO and NCO products from sgs1-mn are severely repressed in the absence of Smc6: Same CO and NCO assay as described in Figure 3 (C, D). Southern blotting of samples from synchronous meiotic time courses at 33°C. Xho1/EcoR1 digests, IS-JM/CO/NCO detection. Left: sgs1-mn, Right: sgs1-mn smc6-56. (C) Total JM signals were quantified, subtracted from background and plotted as % of total signal as a function of time in SPM. Continuous lines: JM from (A). Dotted lines: Inter sister JM from (B). Blue: sgs1-mn, red: sgs1-mn smc6-56. (D) CO signals from (B) were quantified, subtracted from background and plotted as % of total signal as a function of time in SPM. Blue: sgs1-mn, red: sgs1-mn smc6-56. (E) NCO signals from (B) were quantified, subtracted from background and plotted as % of total signal as a function of time in SPM. Blue: sgs1-mn, red: sgs1-mn smc6-56. (F) Meiotic progression is unaffected in sgs1-mn smc6-56: Meiotic progression was followed by spindle labeling. n = 200 cells per experiment, continuous lines: monopolar spindles, dotted lines: bipolar spindles. Blue: sgs1-mn, red: sgs1-mn smc6-56. (G) Meiotic nuclear divisions are blocked in sgs1-mn smc6-56 double mutants. Continuous lines: cells containing one DAPI stained nucleus (1n), dashed lines: cells with 2 nuclei, dotted lines: cells with 4 nuclei (4n). Blue: sgs1-mn, red: sgs1-mn smc6-56. (H, I) Shift to permissive temperature during ndt80 release restores nuclear divisions in smc6-56. (H) Upper panels, green: Wild type, lower panels, red: smc6-56. Continuous lines: Monopolar spindles. Dotted lines: bipolar spindles. From left to right: 1st panel: Time course experiment under restrictive conditions in the ndt80-IN strain background in the absence of inducer (estradiol). 2nd panel: shift to permissive temperature (23.5°C), without release (− estradiol). 3rd panel: release (+ estradiol) into permissive temperature (causes short and synchronous burst of bipolar spindles). 4th panel: release (+ estradiol) into restrictive temperature (causes short and synchronous burst of bipolar spindles). (I) % Multi nuclear cells (2n+4n) are plotted against hours in SPM. Upper panels (grey filled circles): SMC6 ndt80-IN, lower panels (red filled circles): smc6-56 ndt80-IN. Left panels: ndt80 release (+ estradiol) into permissive conditions restores divisions in the smc6-56 background. Right panel: release (+ estradiol) into restrictive temperature maintains the block of division.
Figure 6.
The Smc5/6-Mms21 complex supports the anti-recombinogenic function of Sgs1.
(A) Lack of the Mms21 SUMO ligase mildly compromises chromatin separation in the sgs1-mn background. Left column on green background: From top down: Percent of tetrads with 0, 1,2,3,4 spores containing nuclear DNA. Bottom: Spore viability from tetrad dissection. Right upper panel: Meiotic progression of sgs1-mn mms21-11 was followed by spindle labeling. n = 200 cells, continuous line: monopolar spindles, dotted line: bipolar spindles. Right lower panel: Continuous line: cells containing one DAPI stained nucleus (1n), dotted lines: cells with 2 or 4 nuclei (2n+4n), dashed lines: cells with 4 nuclei (4n). (B) As in (A). The Mms21 SUMO ligase guarantees complete chromatin separation in the zip1Δ mutant indicating that the Mms21 SUMO ligase is required for full Smc5/6-Mms21 resolvase activity. Top to bottom: Percent of tetrads with 0,1,2,3,4 spores containing nuclear DNA. Number at the bottom: Spore viability from tetrad dissection given as percentage. Genotypes indicated above each column. Right upper panel: Meiotic progression of zip1Δ was followed by spindle labeling. n = 200 cells, continuous line: monopolar spindles, dotted line: bipolar spindles. Right lower panel: Meiotic progression of zip1Δ mms21-11 was followed by spindle labeling. n = 200 cells, continuous line: monopolar spindles, dotted line: bipolar spindles. (C; and D) Chromosomal interaction sites and intensities are highly similar between Sgs1 and Smc6. DNA-interaction sites for Sgs1-myc18 (t = 4.5 hours in SPM) and Smc6-myc13 (t = 3.5 hours in SPM) on a 130 kb region of chromosome V. ChIP-seq signals are shown after smoothing (bandwidth: 500 bp), NCIS normalization and background subtraction as described in Materials and Methods. Black: Sgs1. Smc6 signals were plotted in red on the negative scale to facilitate comparison. The obvious Sgs1/Smc6 symmetry in the example region is corroborated genome-wide by the high Pearson correlation (pcorr = 0.8) over more than 6000 peaks per profile. Yellow lollipops: DSB positions as defined in [53] (corresponding to ∼7000 DSB-hotspots across the genome). These positions map overwhelmingly often precisely at the ChIP-seq peaks. The filled grey profile represents Mer2-HAint. (t = 4h) to illustrate core site signals. Note that smaller Sgs1 signals, and to a lesser extent also Smc6 signals also overlap with core sites, as defined by Mer2. (D) Pearson correlation coefficient matrix between the peaks of all profiles shown in this work. Matching peaks (after smoothing to a bandwidth of 300 bp and subtracting the smoothed untagged control) were identified (based on exceeding a certain threshold, set here to Q = 0.7) and their intensities compared by Pearson correlation. The coloring should facilitate comparisons: dark blue represents low correlations whereas lighter shades indicate increased correlation. Intensities between DSBs and ChIP-seq profiles do not match (pcorr<.3), whereas intensities of peaks between Sgs1 and Smc6 profiles usually highly (frequently pcorr>.75).
Figure 7.
Model of how Smc5/6-Mms21 may antagonize inappropriate JMs in meiotic recombination.
(A) A representation of homologous recombination and intermediate pathway choice in the course of budding yeast meiosis is shown, along with the points of function for Smc5/6-Mms21 in antagonizing inappropriate JMs. Meiotic DSBs can engage for two alternative fates: non-ZMM DSBs and ZMM-DSBs (highlighted in green). Regular pathway progression is marked with blue arrows and aberrant deviations are depicted in red. By default, stable strand invasions are disassembled for all breaks through the action of Sgs1 helicase and Smc5/6-Mms21. Non-ZMM DSBs are consequently repaired by SDSA into non-crossovers. In contrast, ZMM-DSBs are in addition also prevented from being repaired at all until licensed in the ZMM pathway for progression to stable invasions, here referred to as “ZMM-Repair Barrier”. When a ZMM-DSB is allowed to form stable SEIs and dHJs, the ZMM-pathway protects that DSB from the action of Sgs1 and Smc5/6-Mms21 and marks the resulting dHJ for CO specific resolution by Exo1-Mlh1/3. Intermediates that deviate from their supposed repair pathways by inappropriate intermediate stabilization and absence of a ZMM label result in “rogue” JMs that depend on Smc5/6-Mms21 for resolution by the rogue JM resolvase Mus81-Mms4 (and probably also Slx1-Slx4 and Yen1). (B) Our data implicates the Smc5/6-Mms21 complex in at least two independent mechanisms for rogue JM avoidance: On one hand prevention of JM formation through Mms21 SUMO E3 mediated regulation of anti-recombinogenic helicases, and on the other hand, the promotion of resolution of JMs by rogue JM resolvases. We also assume that through regulation of helicases, the Smc5/6-Mms21 complex may very likely also be involved in regulating dHJ dissolution by Sgs1-Rmi1-Top3. (C) Smc5/6-Mms21 may survey the DNA for displaced ssDNA at D-loops or HJs by topologically entrapping dsDNA as a sliding SMC ring and binding stably to ssDNA (e.g. at the hinge or any other part of the ring) upon encounter. Stable binding to such ssDNA will constrain D-loop extension and HJ branch migration and label sites with a ssDNA/dsDNA interface where appropriate action of anti-recombinogenic helicases (like Sgs1 or Mph1) and resolvases will be required.