Meiotic recombination intermediates are resolved with minimal crossover formation during return-to-growth, an analogue of the mitotic cell cycle

Abstract

Accurate segregation of homologous chromosomes of different parental origin (homologs) during the first division of meiosis (meiosis I) requires inter-homolog crossovers (COs). These are produced at the end of meiosis I prophase, when recombination intermediates that contain Holliday junctions (joint molecules, JMs) are resolved, predominantly as COs. JM resolution during the mitotic cell cycle is less well understood, mainly due to low levels of inter-homolog JMs. To compare JM resolution during meiosis and the mitotic cell cycle, we used a unique feature of Saccharomyces cerevisiae, return to growth (RTG), where cells undergoing meiosis can be returned to the mitotic cell cycle by a nutritional shift. By performing RTG with ndt80 mutants, which arrest in meiosis I prophase with high levels of interhomolog JMs, we could readily monitor JM resolution during the first cell division of RTG genetically and, for the first time, at the molecular level. In contrast to meiosis, where most JMs resolve as COs, most JMs were resolved during the first 1.5-2 hr after RTG without producing COs. Subsequent resolution of the remaining JMs produced COs, and this CO production required the Mus81/Mms4 structure-selective endonuclease. RTG in sgs1-ΔC795 mutants, which lack the helicase and Holliday junction-binding domains of this BLM homolog, led to a substantial delay in JM resolution; and subsequent JM resolution produced both COs and NCOs. Based on these findings, we suggest that most JMs are resolved during the mitotic cell cycle by dissolution, an Sgs1 helicase-dependent process that produces only NCOs. JMs that escape dissolution are mostly resolved by Mus81/Mms4-dependent cleavage that produces both COs and NCOs in a relatively unbiased manner. Thus, in contrast to meiosis, where JM resolution is heavily biased towards COs, JM resolution during RTG minimizes CO formation, thus maintaining genome integrity and minimizing loss of heterozygosity.

Figure 1. Cell cycle progression and SC breakdown after RTG.

a. Representative images of ndt80 cells (MJL3430) at various stages of RTG, visualized by differential interference contrast (DIC) or by DAPI-staining to detect nuclei (DNA). Note that the daughter cell is elongated as compared to the round mother cell. Scale bar—4µm. b. Time of bud emergence and nuclear division after RTG using SPO11 ndt80Δ (MJL3164, top) or spo11-Y135F ndt80Δ (MJL2807, bottom); the latter do not form SC or JMs. Circles – unbudded cells; squares – cells with a bud and one nucleus; triangles – cells that are undergoing or have finished nuclear division. Values for MJL3164 are from 4 independent determinations. c. SC breakdown upon RTG. Nuclei (MJL3163) were surface-spread and probed with anti-Zip1 antisera. Representative images of nuclei classified as full SC (long, continuous Zip1 lines), partial SC (discontinuous or dotty Zip1) and no SC (no Zip1 chromosomal staining) are shown together with DNA staining. Extrachromosomal Zip1 aggregates (polycomplex) were also detected as a bright-staining body. Scale bar—4 µm. d. Time of SC breakdown after RTG (MJL3163). At least 150 nuclei were scored for each time point. Circles – nuclei with full SC; squares – nuclei with partial SC; triangles – nuclei with no SC. Values are from a single experiment.

Figure 2. The first cell division after RTG involves equational chromosome segregation without replication.

a. Outcome of different types of chromosome segregation after RTG. One homolog is shown as solid line and the other as dashed line. Black and diagonal hatched boxes indicate dominant TRP1 and recessive trp1 alleles, respectively. Reductional chromosome segregation (left) separates homologs, producing a sectored colony with TRP1/TRP1 and trp1/trp1 cells. Equational chromosome segregation (right) separates sister chromatids, producing homogenous TRP1/trp1 colonies. b. Meiotic cells (MJL3163) were plated on YPD, inducing RTG, and 2767 colonies were replica-plated to medium lacking tryptophan. The single Trp⁺/Trp⁻ colony observed is shown. c. Expected outcomes if DNA replication occurs or does not occur before the first nuclear division after RTG. A strain hemizygous for a CEN5-GFP array (black rectangles, see text for details) is illustrated. After 7 hr in meiosis, each cell includes two copies of CEN5-GFP (middle). Replication followed by equational chromosome segregation (left) results in two copies of CEN5-GFP in each cell. Equational chromosome segregation without prior replication (right) leaves a single copy of CEN5-GFP in each cell. d. Upper panel—post-mitotic cells with a hemizygous CEN5-GFP array (MJL3312), from a sample taken 3.5 hr after RTG. All 282 post-mitotic G1 cells examined had a single GFP spot. Lower panel—control cells with a homozygous CEN5-GFP array (MJL3313) growing vegetatively in YPD. An unbudded cell in G1 is shown. 28/104 G1 cells had two GFP dots. Left—Nuclei detected by DNA/DAPI fluorescence; right—GFP fluorescence.

Figure 3. Few COs are produced after RTG.

a. CO detection after RTG. Chromosome VII homologs are shown as solid and dashed lines. Black and grey boxes indicate dominant CYH2 and recessive cyh2-z cycloheximide sensitive and resistant alleles, respectively. If a CO occurs between CYH2 and the centromere, equational chromosome segregation produces either a colony that is uniformly CYH2/cyh2-z (cycloheximide-sensitive), or a colony with a CYH2/CYH2 (cycloheximide-sensitive) sector and a cyh2-z/cyh2-z (cycloheximide-resistant) sector. b. Experimental design. ndt80Δ CDC5-IN (MJL3267) cells are incubated in sporulation medium for 7 hr to uniform pachytene arrest, and aliquots are plated on YPD for RTG (c and d). The culture is then incubated for an additional 4 hr without CDC5 induction and plated on YPD (e), or the culture is incubated for 4hr in the presence of estradiol to induce CDC5 expression before plating on YPD (f). Colonies on YPD are replica-plated to YPD + cycloheximide to detect cyh2-z/cyh2-z recombinants. c, d. Control aliquots plated directly on YPD before replica-plating to YPD + cycloheximide. e. Pachytene-arrested cells were incubated for 4 hr without CDC5 induction before plating on YPD. f. Pachytene-arrested cells were incubated for 4 hr with estradiol to induce CDC5 expression before plating on YPD. Note the marked increase in the frequency of cycloheximide-resistant segregants.

Figure 4. JM resolution and recombinant product formation during meiosis and RTG.

a. Experimental design. Cells with an estrogen-inducible NDT80 allele (MJL3430) are incubated in sporulation medium for 7 hr to uniform pachytene arrest. Estradiol (ED) is added to half of the culture to induce NDT80 expression and the completion of meiosis, while the other half is transferred to YPD to undergo RTG in the absence of NDT80 expression. b. Western blot showing Ndt80 production after addition of ED (meiosis) or after RTG. Arp7 is used as a loading control. Relative Ndt80 levels (arbitrary units) are shown below each lane. c. Western blot showing production of Ndt80-regulated polo-like kinase, Cdc5, and of the G2/M cyclin, Clb2, which is not expressed during meiosis. Arp7 is used as loading control. Relative protein levels (arbitrary units) are shown below each lane. d. Meiotic progression after NDT80 induction by ED addition. The percentage of cells completing meiosis I in a single experiment was determined by DAPI staining and counting the fraction of cells with more than one nucleus (MI + MII). Values are from a single experiment. e. Cell cycle progression after RTG. Cell cycle events were scored as in Figure 1. Values are from three independent experiments. f. Recombination reporter system used to detect recombination intermediates and products . A 3.5 Kb insert with the URA3 (grey) and ARG4 (black) genes is inserted at LEU2 (red) on one chromosome III homolog and at HIS4 (blue), 16.7 Kb away, on the other. 65 nt of yeast telomere sequences (open box), inserted between URA3 and ARG4, create a strong meiotic DSB site (vertical arrow). A short palindrome containing an EcoRI site (lollipop) ∼0.6 kb from the DSB site, creates the arg4-pal allele in the insert at his4. Arrows denote the direction of transcription. Restrictions sites: Xm—XmnI; X—XhoI; E—EcoRI. An XmnI digest probed with ARG4 sequences (black bar) detects dHJ-JMs. A XhoI digest probed with the same sequences detects CO products. An EcoRI/XhoI double digest, probed with HIS4 sequences (blue bar) detects NCO events where the arg4-pal allele is converted to ARG4 (full conversion shown), as well as a subset of COs (CO). It should be noted that a subset of NCOs are detected by this assay. Based on tetrad data from similar strains , we estimate that about 1/6 of total NCOs are detected. g–i. DNA was prepared from NDT80-IN cells (MJL3430) that were either induced to complete meiosis by ED addition or shifted to YPD to undergo RTG, as illustrated in a. Samples were analyzed for JMs, COs and NCOs as illustrated in f. Values for meiosis are from a single experiment; values for RTG are from three independent experiments (for JMs and COs) and two independent experiments for NCOs. g. JM intermediates. Left: blots of XmnI digests probed with ARG4 sequences. In addition to dHJ-JMs, JMs containing 3 or 4 chromatids (multichromatid, mc-JMs) were detected at low levels. Right: frequencies of all JMs, plotted as a percent of total lane signal. h. COs. Left: blots of XhoI digests probed with ARG4 sequences. Right: CO product 2 (CO2) plotted as a percent of total lane signal. i. Noncrossover recombinants. Left: blots of XhoI/EcoRI digests probed with HIS4 sequences. Right: NCOs, plotted as a percent of total lane signal.

Figure 5. Efficient JM resolution without CO production after RTG in the absence of Mus81.

a. Cell cycle progression of ndt80Δ mus81Δ cells (MJL3389) after RTG. Cell cycle events were scored as in Figure 1. b. JM intermediates. Left: blot of XmnI digests probed with ARG4 sequences as in Figure 4. Right: total JMs plotted as a percentage of total lane signal. c. COs. Left: blot of XhoI digests probed with ARG4 sequences, as in Figure 4. Right: CO product 2 (CO2), plotted as a percentage of total lane signal. d. NCOs. Left: blots of XhoI/EcoRI digests probed with HIS4 sequences, as in Figure 4. Right: NCO products plotted as a percentage of total lane signal.

Figure 6. Delayed JM resolution and increased CO formation after RTG in the absence of the Sgs1 helicase.

a. Delayed nuclear division during RTG of in the absence of Sgs1 helicase activity is due to meiotic recombination. Panels show cell cycle progression of ndt80Δ sgs1-ΔC795 cells that are meiotic recombination competent (SPO11, left; MJL3388) or recombination null (spo11, right; MJL3428). b. Joint molecule intermediates. Left: blots of XmnI digests probed with ARG4 sequences. Right: frequencies of total JMs (multichromatid JMs, mcJMs plus dHJ-JMs, filled circles) and of dHJ intermediates (dHJ; empty circles) plotted as a percentage of total lane signal. c. Crossovers. Left: blots of XhoI digests probed with ARG4 sequences. Right: CO product 2 (CO2) are plotted as a percentage of total lane signal. d. Noncrossovers. Left: blots of XhoI/EcoRI digests probed with HIS4 sequences. Right: NCOs, plotted as a percentage of total lane signal.

Figure 7. Modes of dHJ-JM resolution and summary of data.

a. Resolution by junction cleavage . Cleavage of both Holliday junctions in the same orientation (black arrows) yields noncrossovers; cleavage of the two junctions in orthogonal orientations (black and grey arrows) yields crossovers. For simplicity, only one of the two patterns for each type of cleavage is shown. b. Resolution by dissolution , . Helicase-driven convergent junction branch migration, coupled with topoisomerase-removal of superhelical stress, produces only noncrossovers. c. Resolution by replication produces only noncrossovers. d. Summary of JM resolution during RTG. Maximum JM levels in each individual experiment (3 for wild-type, 2 for sgs1-ΔC795 and mus81Δ) were set to 1. For sgs1-ΔC795, 2-chromatid JM values were used, although similar results are obtained with total JMs (2-chromatid + multichromatid). Plotted values represent averages; error bars indicate standard error of the mean. e. Net CO production during RTG. CO levels at 0 hr (the time of RTG) were subtracted from each time-point value and plotted as in d. f. Net NCO production during RTG. NCO levels at 0 hr (the time of RTG) were subtracted from each time-point value and plotted as in d.