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. 2012 Apr 13;149(2):334-47.
doi: 10.1016/j.cell.2012.03.023.

Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase

Kseniya Zakharyevich  1 , Shangming Tang, Yunmei Ma, Neil Hunter
Affiliations

Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase

Kseniya Zakharyevich et al. Cell. .

Abstract

At the final step of homologous recombination, Holliday junction-containing joint molecules (JMs) are resolved to form crossover or noncrossover products. The enzymes responsible for JM resolution in vivo remain uncertain, but three distinct endonucleases capable of resolving JMs in vitro have been identified: Mus81-Mms4(EME1), Slx1-Slx4(BTBD12), and Yen1(GEN1). Using physical monitoring of recombination during budding yeast meiosis, we show that all three endonucleases are capable of promoting JM resolution in vivo. However, in mms4 slx4 yen1 triple mutants, JM resolution and crossing over occur efficiently. Paradoxically, crossing over in this background is strongly dependent on the Blooms helicase ortholog Sgs1, a component of a well-characterized anticrossover activity. Sgs1-dependent crossing over, but not JM resolution per se, also requires XPG family nuclease Exo1 and the MutLγ complex Mlh1-Mlh3. Thus, Sgs1, Exo1, and MutLγ together define a previously undescribed meiotic JM resolution pathway that produces the majority of crossovers in budding yeast and, by inference, in mammals.

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Figures

Figure 1
Figure 1. Physical Assay System for Monitoring Meiotic Recombination
(A) Map of the HIS4LEU4 locus showing diagnostic restriction sites and position of the probe. DNA species detected with Probe 4 are shown below. X, XhoI; B, BamHI; N, NgoMIV; mc-JM, multi-chromatid joint molecule; IS-JM, intersister joint molecule; IH-JM, interhomolog joint molecule; SEI, Single-End Invasion, DSB, double-strand break. (B) 1D gel analysis of crossovers and DSBs. Southern image of XhoI-digested genomic DNA hybridized with Probe 4. (C) Inferred structures of SEI and dHJ intermediates. (D) 2D gel analysis of JMs. Southern image of a native/native 2D gel. Species detailed in (A) are highlighted. (E) Noncrossover analysis. Southern image of XhoI + NgoMIV double digested genomic DNA showing diagnostic bands. Note that this assay detects a representative subset of total recombinants.
Figure 2
Figure 2. Survey Of Candidate JM Resolving Enzymes
(A) Representative Southern images of native/native 2D gels showing JMs at 24 hours after transfer into the sporulation media in various triple mutant strains. (B) Quantitative analysis of joint molecules. % DNA is percent of total hybridization signal. Total JM levels ± SEM at 24 hours are shown. (C) Representative Southern images of 1D gel crossover analysis. (D) Quantitative analysis of crossover products after 24 hrs. Error bars show SEM.
Figure 3
Figure 3. Recombination Is Efficient In The Triple Resolvase Mutant, mms4 slx4 yen1
(A) Representative cells from wild-type and mms4 slx4 yen1 strains. Brightfield and DAPI-stained images of the same cells are shown. (B) Southern images of noncrossover analysis. (C) Noncrossover levels at 24 hrs. (D) Southern images of crossover analysis. (E) Crossover levels at 24 hrs. (F) Southern images of JM analysis. (G) JM levels at 24 hrs. Error bars in all panels show SEM.
Figure 4
Figure 4. Yen1 an Slx1–Slx4 Are Cryptic Resolvases
(A) Representative Southern images of crossover analysis in wild-type, yen1, slx1 and slx4 strains. (B) 2D analysis of JMs. Southern images of representative 2D gels are shown. (C) Quantitative analysis of DSBs, JMs, crossovers and meiotic divisions (MI±MII). MI±MII is percent of cells that have completed either first or second meiotic divisions as detected by the number of DAPI-staining bodies. (D) Genetic analysis of crossing-over. A map of the two intervals flanking the HIS4LEU2 locus on yeast chromosome III is shown. Graphs show genetic distances ± SE. cM, centi-Morgans. (E) Spore viability of the indicated strains. At least 100 tetrads were dissected in each case. (F) Epistasis analysis of yen1 and other resolvase mutants. Graph shows crossover levels ± SEM at 24 hrs measured by physical analysis at HIS4LEU2. (G) Epistasis analysis of slx4 and other resolvase mutants. Graph shows crossover levels ± SEM at 24 hrs measured by physical analysis at HIS4LEU2. (H) Representative cells from wild-type, mms4 yen1, sgs1 slx1 and sgs1 slx4 strains. Brightfield and DAPI-stained images of the same cells are shown. (I) 2D analysis of JMs in mms4 yen1 and sgs1 slx4 at 24 hrs. Southern images of representative 2D gels are shown. (J) JM levels in wild-type, mms4 yen1 and sgs1 slx4 at 24 hrs. Error bars show SEM.
Figure 5
Figure 5. Sgs1 and Exo1-MutLγ Promote JM Resolution And Crossing-Over
(A) Representative Southern images of 1D gel crossover analysis in wild-type, mms4 slx4 yen1, mms4 slx4 yen1 sgs1, mms4 slx4 yen1 mlh3 and mms4 slx4 yen1 mlh3-D523N cells. (B) Crossover levels at 24 hrs. (C) Representative Southern images of noncrossover analysis. (D) Noncrossover levels at 24 hrs. (E) 2D analysis of JMs. Representative 2D panels are shown. (F) JM levels at 24 hrs. (G) Representative Southern images of crossover analysis in wild-type, exo1, mms4 yen1, and exo1 mms4 yen1 cells. (H) Crossover levels at 24 hrs. In all panels, error bars show SEM.
Figure 6
Figure 6. JM Resolution And Crossing-Over Is Abolished In An mlh3 mms4 slx4 sgs1 yen1 Quintuple Mutant
(A) Representative Southern images of crossover analysis in wild-type and mlh3 mms4 slx4 sgs1 yen1 quintuple mutant cells. (B) Crossover levels at 24 hrs. (C) Representative Southern images of noncrossover analysis in wild-type and mlh3 mms4 slx4 sgs1 yen1 quintuple mutant cells. (D) Noncrossover levels at 24 hrs. (E) 2D analysis of joint molecules in wild-type and mlh3 mms4 slx4 sgs1 yen1 quintuple mutant cells. Representative 2D panels are shown. (F) JM levels at 24 hrs. The first bar shows data for wild type. In all panels, error bars show SEM.
Figure 7
Figure 7. Model Of Joint Molecule Resolution During Meiosis
A summary of meiotic JM metabolism is shown for budding yeast and, by extension, other organisms that rely primarily on the MutLγ crossover pathway, such as mammals and plants. Red and black lines distinguish parental homologs. Sister chromatids are also present at this stage but are not shown. Dashed lines indicate newly synthesized DNA. Initial ZMM-dependent stabilization of strand-exchange intermediates may occur at most or all recombination sites in order to facilitate or stabilize homolog synapsis (as suggested by the synapsis defects of msh4 and msh5 mutants and the large numbers of MutSγ foci observed during the zygotene and early pachytene stages in several organisms)(Edelmann et al., 1999; Higgins et al., 2008b; Kneitz et al., 2000; Moens et al., 2002). BLM colocalizes with MutSγ at this stage (Holloway et al., 2010; Moens et al., 2002) and, as suggested above, could facilitate initial JM stabilization and regulate the transition to dHJs. Sgs1/BLM also limits formation of aberrant JMs that may require processing by the structure selective nucleases, mainly Mus81-Mms4 (cryptic activities of Slx1–Slx4 and Yen1 are indicated by parentheses). Sgs1 could limit aberrant JMs by unwinding D-loops and/or dissociating nascent dHJs that result from promiscuous strand-exchange (Oh et al., 2007). Continued stabilization of JMs occurs only at designated crossover sites and leads to formation of dHJs. Exo1-MutLγ is then assembled at these sites and activated to cleave dHJs asymmetrically to produce crossovers. These “class I” crossovers show a regulated distribution with respect to crossover assurance (≥1 per homolog pair) and crossover interference (adjacent crossovers are widely and evenly spaced)(Berchowitz and Copenhaver, 2010). Asymmetric loading of MutSγ or some other polarity signal may direct crossover-specific resolution via Exo1-MutLγ. Absence of continued JM stabilization at noncrossover precursors results in disassembly via Sgs1 helicase action (and perhaps other helicases) to promote synthesis-dependent strand-annealing.
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