Genetics: Early Online, published on May 16, 2016 as 10.1534/genetics.115.182923
Synaptonemal Complex Proteins of Budding Yeast Define Reciprocal Roles in MutSγMediated Crossover Formation
Karen Voelkel-Meiman, Shun-Yun Cheng, Savannah J. Morehouse and Amy J. MacQueen1
Department of Molecular Biology and Biochemistry
Wesleyan University, Middletown, CT, United States of America
1
Corresponding author:
Phone: (860) 685-2561
Fax: (860) 685-2141
E-mail: [email protected]
Running Title: SC Proteins and Crossover Recombination
Copyright 2016.
1
Abstract
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During meiosis, crossover recombination creates attachments between homologous chromosomes that
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are essential for a precise reduction in chromosome ploidy. Many of the events that ultimately process
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DNA repair intermediates into crossovers during meiosis occur within the context of homologous
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chromosomes that are tightly aligned via a conserved structure called the synaptonemal complex (SC),
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but the functional relationship between SC and crossover recombination remains obscure. There exists
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a widespread correlation across organisms between the presence of SC proteins and successful
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crossing over, indicating that the SC or its building block components are pro-crossover factors. For
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example, budding yeast mutants missing the SC transverse filament component, Zip1, and mutant cells
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missing the Zip4 protein, which is required for the elaboration of SC, fail to form MutSγ-mediated
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crossovers. Here we report the reciprocal phenotype - an increase in MutSγ-mediated crossovers
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during meiosis – in budding yeast mutants devoid of the SC central element components Ecm11 or
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Gmc2, and in mutants expressing a version of Zip1 missing most of its N terminus. This novel
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phenotypic class of SC-deficient mutants demonstrates unequivocally that the tripartite SC structure is
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dispensable for MutSγ-mediated crossover recombination in budding yeast. The excess crossovers
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observed in SC central element-deficient mutants are Msh4-, Zip1-, and Zip4-dependent, clearly
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indicating the existence of two classes of SC proteins - a class with pro-crossover function(s) that are
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also necessary for SC assembly, and a class that is not required for crossover formation but essential
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for SC assembly. The latter class directly or indirectly limits MutSγ-mediated crossovers along meiotic
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chromosomes. Our findings illustrate how reciprocal roles in crossover recombination can be
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simultaneously linked to the SC structure.
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Introduction
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The synaptonemal complex (SC) is correlated with successful interhomolog crossover formation
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during meiosis; mutants missing SC components nearly always exhibit a decrease in crossovers and
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(as a consequence) increased errors in chromosome segregation at meiosis I (PAGE and HAWLEY
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2004). Transverse filaments establish a prominent component of the typically tripartite SC
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structure; transverse filaments are comprised of coiled coil proteins that form rod-like entities that
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orient perpendicular to the long axis of aligned chromosomes, bridging chromosome axes at a
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distance of approximately 100 nanometers along the entire length of the chromosome pair (PAGE
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and HAWLEY 2004). The largely coiled-coil Zip1 protein is a major (and perhaps the only)
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transverse filament protein of the budding yeast SC (DONG and ROEDER 2000; SYM et al. 1993)
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(Figure 1A).
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Budding yeast mutants that are missing the SC transverse filament protein Zip1 lack MutSγ-
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mediated crossovers (BORNER et al. 2004; NOVAK et al. 2001; VOELKEL-MEIMAN et al. 2015).
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Furthermore, crossover levels in double mutants missing Zip1 and any of the so-called Synapsis
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Initiation Complex (SIC) proteins (Zip2, Zip3, Zip4 and Spo16), which are required for SC
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assembly, and in triple mutants that simultaneously lack Zip1, Zip4 and/or Msh4, indicate that SIC
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proteins promote the same (MutSγ-mediated) set of crossovers attributed to Zip1 function (BORNER
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et al. 2004; NOVAK et al. 2001; SHINOHARA et al. 2008; TSUBOUCHI et al. 2006; VOELKEL-MEIMAN
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et al. 2015, this work).
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One exception to the strong positive correlation between SC proteins and crossover formation in
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budding yeast is our prior observation of elevated crossover recombination in SUMO-deficient
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mutants, which also exhibit diminished tripartite SC assembly (synapsis) (VOELKEL-MEIMAN et al.
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2013). Because SUMOylation is associated with a variety of molecular targets and because mutants
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missing the SUMOylated protein Ecm11 (a structural component of the budding yeast SC central
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element) were reported to exhibit reduced meiotic crossovers (HUMPHRYES et al. 2013), the
2
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observation of increased crossovers in SUMO-deficient mutants was not interpreted at the time as
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evidence that the budding yeast SC has an antagonistic relationship with meiotic crossover
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formation.
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The tight correlation between defects in synapsis and crossing over suggests the possibility that
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the SC structure itself has a functional role in meiotic crossover recombination. The maturation of
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recombination intermediates occurs largely within the context of assembled SC, but how the SC
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structure interfaces with the double strand break (DSB) repair process remains obscure. In budding
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yeast it is thought that at least some SC proteins facilitate early steps in interhomolog recombination
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that may occur prior to the elaboration of full-length SC (BORNER et al. 2004; HUNTER and
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KLECKNER 2001; STORLAZZI et al. 1996) leaving open the question of whether the mature SC is
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required at all for crossover formation. Recent genetic data from C. elegans and rice, on the other
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hand, have raised the paradox that while SC components are essential for meiotic crossovers, strains
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partially depleted for SC protein activity exhibit an increase in crossovers (LIBUDA et al. 2013;
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WANG et al. 2015). These observations indicate that SC proteins are associated with both positive
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and negative roles in crossing over, but it remains unknown how the pro- and anti- crossover
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functions attributed to SC components in these organisms are related to one another at the
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molecular level.
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Here we describe a set of SC-deficient budding yeast mutants with a novel phenotype that
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cleanly uncouples SC-associated crossover recombination from tripartite SC assembly. We find that
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structural components of the budding yeast SC can be classified into two groups based on their
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reciprocal affects on crossover formation: Mutants missing building blocks of the SC central
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element, Ecm11 or Gmc2 (HUMPHRYES et al. 2013; VOELKEL-MEIMAN et al. 2013), and strains
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expressing a version of Zip1 that is missing most of its N terminus (the zip1-N1 mutant allele;
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(TUNG and ROEDER 1998)), do not exhibit the deficiency in crossing over characteristic of
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previously described synapsis-deficient mutants. Instead, ecm11, gmc2, and zip1-N1 mutants
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display an increase in MutSγ-mediated crossing over. Our findings unequivocally demonstrate that
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the tripartite SC structure is dispensable for “pro” crossover recombination functions in budding
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yeast, and these data furthermore suggest that elaborated SC structure directly or indirectly limits
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the formation of MutSγ-mediated interhomolog crossovers during meiosis.
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Methods
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Strains and genetic analysis
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Yeast strains used in this study are isogenic to BR1919-8B (ROCKMILL and ROEDER 1998; Table
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S5) and were generated using conventional crossing and genetic manipulation procedures. Two
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distinct sets of markers were used for tetrad analysis experiments shown in Table 1 and Table 2.
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Both strains carry an hphMX4 cassette inserted near the chromosome III centromere, ADE2 inserted
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upstream of the RAD18 locus, a natMX4 cassette inserted near the HMR locus, TRP1MX4 inserted
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62bp downstream of the SPO11 locus (KEE and KEENEY 2002) and URA3 replacing SPO13. In
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strains linked to Table 1, LYS2 was inserted on chromosome VIII at coordinate 210,400bp. In
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strains linked to Table 2, LEU2 and THR1 were inserted on chromosome XI at chromosomal
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coordinates 152,000 and 193,424bp, respectively. Tetrad analysis, crossover interference analyses,
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and prototroph experiments were carried out on solid media, as previously described (VOELKEL-
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MEIMAN et al. 2015). All statistical analyses were performed using GraphPad InStat software.
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Physical assays, pulsed field gel electrophoresis, and Southern blotting
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Agarose plugs were prepared from meiotic cultures at 0, 40 and 70 hours of sporulation and
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subjected to pulsed-field gel analysis. For Southern blotting, a 1 kb probe from the THR4 region of
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chromosome III was prepared using a DIG High Prime DNA Labeling and Detection Kit (Roche).
4
96
A Syngene “G:Box” was used to detect chemiluminescence and the Syngene “Gene-Tools”
97
program was used to analyze the data. A value for % recombination was calculated by summing
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twice the intensity of the trimer band (a double crossover product) plus the dimer band (product of a
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single crossover) over the total intensity of the three bands (trimer, dimer and monomer). Note that
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circular chromosome III chromatids do not enter the gel, and thus are not included in the calculation
101
to estimate recombination. The average of two experiments is presented.
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Western blot
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Protein pellets were isolated from 5 mL of sporulating cell culture by TCA precipitation as in
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(HOOKER and ROEDER 2006). The final protein pellet was suspended in 2× Laemmli sample buffer
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supplemented with 30 mM DTT at a concentration of ∼10 µg/µl. Protein samples were heated for
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10 minutes at 65°, centrifuged at top speed and ∼100 µg was loaded onto an 8%
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polyacrylamide/SDS gel. PDVF membranes were prepared according to manufacturer's (Bio-Rad)
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recommendation, equilibrating with Towbin buffer for 15 minutes after methanol wetting. Transfer
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of proteins to PDVF membranes was done following Bio-Rad Protein Blotting Guide for tank
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blotting using Towbin Buffer; stir bar and ice pack were used, transfer was done at 60 V for 1 hour.
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Ponceau S was used to detect relative protein levels on the PVDF membrane after transfer. Mouse
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anti-MYC (9E10, Invitrogen) was used at 1:2500. Incubations with primary antibody were
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performed overnight at 4°C. HRP-conjugated AffiniPure goat anti-mouse antibody
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(JacksonImmunoResearch) was used at 1∶5000 in TBS-T for 1 hour at RT. Amersham ECL Prime
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Western Blotting Detection Reagent was used to visualize antibodies on the membranes; a Syngene
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G:Box and the Syngene GeneTools program was used to detect and analyze the data.
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Cytological analysis and imaging
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Meiotic chromosome spreads, staining and imaging were carried out as previously described
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(ROCKMILL 2009) with the following modifications: 80 µl 1xMES and 200 µl 4%
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paraformaldehyde fix were added to spheroplasted, washed cells, then 80 µl of resuspended cell
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solution was put directly onto a frosted slide and cells were distributed over the entire slide using
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the edge of a coverslip with moderate pressure. The slide was allowed to air dry until less than half
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of the liquid remained, and then washed in 0.4% Photo-flo as described. The following primary
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antibodies were used: mouse anti c-MYC (1∶200) (9E10, Invitrogen), affinity purified rabbit anti-
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Zip1 (1:100) (raised at YenZym Antibodies, LLC, against a C terminal fragment of Zip1 as
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described in (SYM et al. 1993)), rat anti-α-tubulin antibody (Santa Cruz). Secondary antibodies
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were obtained from Jackson ImmunoResearch and used at a 1:200 dilution. Imaging was carried out
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using a Deltavision RT imaging system (Applied Precision) adapted to an Olympus (IX71)
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microscope.
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Cells were prepared for multinucleate analysis (Figure S2B) by first transferring them from
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solid sporulation media into cold 50% ethanol, and storing fixed cells at -20° until all time points
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were collected. Next, 1 µL of fixed cells were transferred to a single well of a multi-well slide and
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allowed to dry. Vectashield mounting medium (Vector Laboratories) containing 1 µg/mL DAPI
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was placed on top of the dried cells and a cover slip added.
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Results and Discussion
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Excess interhomolog crossovers form in ecm11 and gmc2 mutants
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In budding yeast and in many other organisms, a “central element” substructure lies at the midline of
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the SC (HAMER et al. 2006; VOELKEL-MEIMAN et al. 2013). SUMOylated and unSUMOylated Ecm11,
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and (by extension) the Ecm11-interacting protein Gmc2, are components of the central element
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substructure, which assembles close to Zip1’s N termini within the mature budding yeast SC
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(HUMPHRYES et al. 2013; VOELKEL-MEIMAN et al. 2013). In stark contrast to the reduced meiotic
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recombination frequencies observed in strains missing any of several other proteins required for SC
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assembly in budding yeast, such as zip1, zip2, zip4 and spo16 mutants (BORNER et al. 2004; CHUA and
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ROEDER 1998; SHINOHARA et al. 2008; SYM and ROEDER 1994; TSUBOUCHI et al. 2006; VOELKEL-
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MEIMAN et al. 2015), we discovered that meiotic interhomolog crossovers are elevated in synapsis
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defective, ecm11 and gmc2 mutants (Figure 1B-C, Table 1). Tetrad analysis was used to measure
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crossover frequency in seven intervals on chromosomes III and VIII. In six out of seven intervals,
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crossovers in ecm11 (null), ecm11[K5R, K101R] (non-SUMOylatable) or gmc2 mutants are elevated
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to 113%-280% of the wild-type level (Figure 1C, Table 1). Thus, unlike the transverse filament
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component Zip1 and other pro-synapsis factors in budding yeast, Ecm11 and Gmc2 are structural
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components of budding yeast SC that are dispensable, per se, for meiotic crossing over.
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We also measured non-Mendelian segregation, a reflection of gene conversion resulting from
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interhomolog recombination (both crossover and noncrossover) events, for every marker included in
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our crossover recombination analysis. Consistent with our observation of an elevation in the number of
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interhomolog crossovers, a four to seven-fold increase in overall gene conversion levels was observed
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in ecm11, ecm11[K5R, K101R], and gmc2 mutants relative to wild type (Table S1). These data
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indicate that both crossover and noncrossover interhomolog recombination events are elevated when
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Ecm11 or Gmc2 is absent.
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The excess crossovers in ecm11 mutants are dependent on MutSγ
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Mutants missing the Msh4 component of MutSγ exhibit 29%-73% of the wild type crossover level,
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depending on the interval examined (Figure 1C, Figure 3C, Table 1, Table 2). The diminished
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crossover phenotype observed in msh4 mutant cells is epistatic to the excess crossover phenotype of
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ecm11 strains: The ecm11 msh4 double mutant exhibits crossover levels that are similar to the low
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levels of the msh4 single mutant (Figure 1C, Table 1). Thus, unlike the excess crossovers observed in
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strains deficient for Sgs1 helicase activity during meiosis (JESSOP et al. 2006), the additional
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crossovers in ecm11 mutants are MutSγ-mediated.
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Under normal circumstances, the resolution of most crossover-designated recombination
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intermediates in budding yeast is dependent on MutLγ (KOLAS and COHEN 2004; ZAKHARYEVICH et
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al. 2012). Removal of the MutLγ component, Mlh3 from ecm11 mutant strains results in a reduced
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number of interhomolog crossovers, although to a lesser extent than ecm11 msh4 double mutants: the
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interhomolog crossover frequency displayed by ecm11 mlh3 double mutants appeared midway
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between the low crossover frequency of msh4 and the high crossover frequency of ecm11 mutant
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strains (Table 1). This observation is consistent with the proposal that MutLγ is not per se essential for
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the resolution of MutSγ intermediates but if present, channels those intermediates in a biased manner
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toward a crossover outcome (DE MUYT et al. 2012; ZAKHARYEVICH et al. 2012). Accordingly, in the
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absence of MutLγ activity, MutSγ crossover-designated intermediates are presumably resolved in an
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unbiased manner by structure-selective nucleases such that they give rise to both crossovers and
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noncrossovers with equal frequency.
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Surprisingly, removal of Mlh3 from ecm11 mutants results in double the frequency of non-
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Mendelian segregation relative to the ecm11 single mutant (Table S1). Given the fact that the
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frequency of gene conversion in the mlh3 single mutant resembles wild-type meiotic cells, the elevated
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frequency in the ecm11 mlh3 double mutant suggests that Mlh3 acts synergistically with Ecm11 in an
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activity that ultimately limits interhomolog recombination.
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The MutSγ-mediated crossovers in ecm11 mutants rely on Zip1- and Zip4 proteins
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Using a physical assay for recombination, we observed that the excess crossovers that occur when
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SC central element protein Ecm11 is absent relies on the SC transverse filament protein, Zip1, as
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well as on the Synapsis Initiation Complex protein, Zip4. The “circle-linear” assay estimates
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crossover frequency based on the relative abundance of crossover chromatid products resulting
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from recombination between circular and linear chromosomes III (GAME et al. 1989; VOELKEL-
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MEIMAN et al. 2015) (See Figure 2 legend). A limitation of the assay, which is relevant to this
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study, is that it underestimates crossover frequency (since chromosomes with more than two
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crossovers are not detectable), and thus likely will not report increases above the wild-type
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crossover frequency. However, the circle-linear assay is a powerful tool for detecting a reduction in
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crossing over, particularly for mutants such as zip1 and zip4 where diminished spore production in
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our strain background precludes tetrad analysis. Using the circle-linear assay, a prior study reported
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a delay and overall reduction in the accumulation of crossovers in ecm11 and gmc2 mutants at time
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points through 48 hours of sporulation (HUMPHRYES et al. 2013). In our analysis of ecm11,
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ecm11[K5R, K101R] and gmc2 mutants using the circle-linear assay, a mild reduction in the
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accumulation of resolved crossover recombination intermediates was observed at 40 hours of
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sporulation, but an approximately wild-type crossover frequency was observed for these mutants at
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70 hours (Figure 2). The wild-type crossover frequency observed in ecm11, ecm11[K5R, K101R],
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and gmc2 mutants at 70 hours is in sharp contrast to the diminished frequency (~30%) measured in
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the SC-deficient zip1 and zip4 mutants at this time point (Figure 2). Our analysis using this assay
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moreover revealed that crossovers diminish to zip1, zip4, msh4, or msh5 single mutant levels when
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Zip1, Zip4, Msh4, and Msh5, respectively, are removed from ecm11 mutant strains (Figure 2). Thus
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the extra crossovers formed in ecm11 mutants (observed by genetic analysis) rely not only on the
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Msh4-Msh5 complex, but on Zip1 and Zip4 proteins as well.
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Altogether, our data reveal that two classes of SC structural proteins exist in budding yeast. The
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SC transverse filament component Zip1 is essential for building tripartite SC and for MutSγ-
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mediated crossover formation, while the central element components Ecm11 and Gmc2 are
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essential for tripartite SC assembly but dispensable for Zip1/Zip4/MutSγ-mediated crossing over.
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While dispensable for crossing over per se, the delayed accumulation of crossovers observed in
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ecm11 and gmc2 mutants (HUMPHRYES et al. 2013) does suggest that Ecm11 and Gmc2 indirectly
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or directly influence the rate that crossovers form, likely through promoting the timely resolution of
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crossover-designated intermediates at the end of prophase (see below).
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A zip1 allele missing N terminal residues exhibits elevated MutSγ-dependent crossing over
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We next identified a zip1 non-null allele that separates Zip1’s role in SC formation from its role in
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mediating MutSγ-dependent recombination. The zip1-N1 allele encodes a protein missing residues
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21-163, corresponding to the majority of N terminal residues prior to Zip1’s extended central
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coiled-coil region (TUNG and ROEDER 1998; Figure 3A). Prior analysis of crossing over within two
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adjacent intervals on chromosome III in zip1-N1 meiotic cells of an SK1 strain background revealed
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an increase in crossover recombination in the CEN3-MAT interval, to 114% of the wild-type level,
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and a ~30% decrease in crossing over in the HIS4-CEN3 interval (TUNG and ROEDER 1998). We
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performed tetrad analysis on zip1-N1 mutants of a BR1919 - derived background (ROCKMILL and
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ROEDER 1998) and found elevated crossing over, corresponding to 115%-228% of wild type levels,
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in five of six genetic intervals representing regions of chromosomes III, VIII and XI (Figure 3C,
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Table 2). Only one interval in zip1-N1 mutants showed a wild-type crossover frequency. Our
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findings demonstrate that at least in the BR1919 background, crossover recombination is elevated
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above the wild-type level in zip1-N1 mutant cells.
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We next explored how the excess crossovers identified in zip1-N1 mutants are related to the
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excess crossovers we observed in ecm11 mutants. Crossover levels in zip1-N1 ecm11 double
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mutants were not dramatically different from either single mutant, indicating that Ecm11 and Zip1-
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N1 proteins interface with the same crossover control pathway. Accordingly, crossover levels are
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reduced in zip1-N1 mutants when either MSH4 or MLH3 activities are absent (Figure 3C, Table 2).
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zip1-N1 mutants display an increase in non-Mendelian segregation at markers on both
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chromosomes III and VIII relative to wild type (Table S1). However, overall gene conversion levels
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(a measure of total interhomolog events) in zip1-N1 strains are approximately half the levels
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observed in ecm11, ecm11[K5R, K101R], and gmc2 mutants, despite the fact that interhomolog
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crossover recombination is increased to similar levels in these mutants (Table 2). Based on this data
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we surmise that a substantial fraction of the excess interhomolog recombination events observed in
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ecm11 and gmc2 mutants are associated with a noncrossover outcome. Interestingly, zip1-N1 is
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epistatic to ecm11 with respect to its gene conversion phenotype, revealing a potential role for Zip1
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in influencing the number of interhomolog noncrossover recombination events that occur when
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Ecm11 is absent.
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The zip1-N1 allele encodes a separation-of-function protein that fails to assemble tripartite SC
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Although the precise molecular relationship between budding yeast transverse filaments and central
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element proteins remains unknown, the Ecm11 and Gmc2 central element proteins localize near Zip1’s
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N termini within the tripartite SC (VOELKEL-MEIMAN et al. 2013). We therefore reasoned that the
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shared phenotype of ecm11, gmc2, and zip1-N1 mutants may be caused by a failure to assemble the
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central element substructure of the tripartite SC. Based on the electron microscopy done in an earlier
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study (TUNG and ROEDER 1998), at least some pachytene stage chromosome axes in zip1-N1 meiotic
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nuclei appeared intimately aligned along their entire lengths, suggesting that normal SC might
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assemble using Zip1-N1 protein as a building block. Importantly, however, this earlier study also
259
found that ~97% of meiotic nuclei at 13, 15, and 17 hours of sporulation exhibited either no Zip1-N1
260
accumulation, or a “dotty” Zip1-N1 distribution pattern on chromosomes (TUNG and ROEDER 1998).
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Based on our observation of elevated crossing over in zip1-N1 strains, we hypothesized that the
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intimate alignment between meiotic chromosome axes in zip1-N1 mutants reflects pseudosynapsis
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arising as a consequence of numerous interhomolog recombination intermediates that promote local
264
points of association along the length of chromosomes (JESSOP et al. 2006), and not from an assembled
265
tripartite SC structure.
266
Indeed, when we analyzed the distribution of Ecm11-MYC and Zip1 proteins on surface-spread
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meiotic chromosomes we discovered that normal SC fails to assemble in zip1-N1 mutants (Figure
268
4A, B). Wild-type meiotic nuclei at the pachytene stage of prophase exhibit completely coincident
269
Zip1 and Ecm11 assembled along the full length of aligned homolog pairs. The coincident labeling
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of Zip1 and Ecm11 reflects the interdependent arrangement of these central element and transverse
271
filament proteins within the higher-order architecture of the wild-type SC (Figure 4A and
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VOELKEL-MEIMAN et al. 2013). In zip1-N1 mutants however, Zip1-N1 and Ecm11 proteins each
273
assemble foci and very short linear stretches, and Zip1-N1 structures do not robustly coincide with
274
Ecm11 assemblies on chromosomes (zoomed insets, Figure 4A). Consistent with an SC assembly
275
defect, Ecm11 SUMOylation, which is required for SC assembly and normally relies to a large
276
extent on Zip1 (HUMPHRYES et al. 2013), is diminished and severely delayed in zip1-N1 mutants
277
(Figure 4C, 4D).
278
Taken together, the shared phenotype of the ecm11, gmc2, and zip1-N1 mutants suggests the
279
possibility that assembly of the SC central element limits MutSγ interhomolog crossover formation. A
280
direct or an indirect mechanism could account for how assembled SC limits interhomolog crossovers,
281
as discussed below.
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Crossover interference is weakened slightly in ecm11, gmc2 and zip1-N1 mutants
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MutSγ-mediated crossovers display positive interference, in that detectable double crossover events
285
in a given chromosomal region occur less frequently than expected based on a random distribution
286
(NISHANT et al. 2010; NOVAK et al. 2001). While SC components are required for the successful
287
generation of interfering (MutSγ) crossovers, other studies have suggested that SC is dispensable
288
for crossover interference (FUNG et al. 2004; ZHANG et al. 2014a; ZHANG et al. 2014b). We used
289
coefficient of coincidence (PAPAZIAN 1952) and interference ratio (MALKOVA et al. 2004) methods
290
to ask whether the SC-independent, MutSγ-mediated crossovers in ecm11 and gmc2 mutants exhibit
291
interference. Wild-type strains displayed robust crossover interference in all intervals using either
292
method (Table 1, Figure S1, Table S2), whereas each method indicated weakened crossover
293
interference in msh4, ecm11, ecm11[K5R, K101R], and gmc2 mutants, although we note that most
294
of the interference measurements for msh4 strains are not statistically significant due to an
295
insufficient number of crossover events. In ecm11, ecm11[K5R, K101R], gmc2, and zip1-N1
296
mutants (where Msh4-mediated crossovers are in excess), NPD(observed)/NPD(expected) ratios
297
appeared as robust as wild-type in some intervals but weaker in others, particularly in the SPO11-
298
SPO13 interval on chromosome VIII.
299
Using the interference ratio method (Figure S1 and Table S2), we found that the presence of a
300
crossover in one interval decreases the likelihood of crossing over in an adjacent interval (exerts
301
positive interference) in wild-type strains. Similar to our coefficient of coincidence measurements,
302
interference as measured by the interference ratio method in ecm11, ecm11[K5R, K101R], gmc2,
303
and zip1-N1 mutant strains appeared weaker than wild type, but not absent, in most interval pairs
304
(Figure S1, Table S2).
305
The presence of (albeit weakened) crossover interference in ecm11, gmc2, and zip1-N1 mutants is
306
consistent with models that propose that SC is not required for interference in budding yeast (FUNG et
307
al. 2004; ZHANG et al. 2014a; ZHANG et al. 2014b).
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The fraction of recombination events that resolve to a crossover outcome is the same or
310
diminished in ecm11 mutant meiotic cells relative to wild type
311
One explanation for the increased number of interhomolog crossover events in ecm11, gmc2, and
312
zip1-N1 mutants is that the number of interhomolog repair intermediates is normal but the absence
313
of central element proteins (or tripartite SC) increases the likelihood that a given interhomolog-
314
engaged repair intermediate is resolved toward a crossover versus a non-crossover outcome. We
315
tested this possibility by measuring the frequency of crossing over associated with meiotic
316
interhomolog recombination events at ARG4 and LEU2 in wild-type and ecm11 strains (Figure 5).
317
Interhomolog recombination events at ARG4 or LEU2 were identified by selecting prototrophs
318
among spore products from diploids carrying arg4 and leu2 heteroalleles; flanking genetic markers
319
were then used to determine the fraction of interhomolog recombination events associated with a
320
crossover. This experiment revealed that the percent of recombination events accompanied by a
321
crossover at either locus is similar to or diminished in ecm11 mutants relative to wild type (51.7%
322
versus 70.4% at ARG4 and 47.9% versus 47.1% at LEU2, respectively; Figure 5). These data
323
suggest that Ecm11’s absence does not increase the likelihood that a given interhomolog
324
recombination intermediate is resolved toward a crossover outcome. Alternatively, the presence of
325
SC central region proteins might act to limit the likelihood that initiated recombination events
326
productively engage with the homolog for repair.
327
A third possibility is that the presence of SC central region proteins may directly or indirectly
328
down-regulate the number of recombination events that are initiated during meiotic prophase. This
329
possibility is supported by the recent demonstration of elevated recombination initiation (Spo11-
330
mediated DNA double strand breaks) in zip1, zip3, zip4 and spo16 mutants, which are missing
331
proteins with both pro-crossover and pro-SC assembly roles (THACKER et al. 2014). If the same
14
332
feedback mechanism that leads to increased Spo11-mediated recombination initiation in zip1, zip3,
333
zip4 and spo16 mutants is responsible for the elevated number of MutSγ-mediated crossovers we
334
observe in ecm11, gmc2 and zip1-N1 mutants, this would suggest the interesting possibility that the
335
feedback mechanism itself is coupled to a deficit in tripartite SC, rather than to a deficit in SIC
336
protein-mediated crossover activity.
337
338
ecm11 and gmc2, but not zip1-N1 mutants, display delayed progression through late prophase
339
While, in principle, the SC may directly prevent recombination initiation or influence how
340
recombination events are processed, we note two alternative models (which are not mutually
341
exclusive) in which the presence of SC central element proteins prevent elevated crossing over
342
indirectly. First, an increase in crossovers might not be the result of absent tripartite SC per se but
343
instead due to a diminished level of a particular SC-associated protein, which has a dual role in SC
344
assembly and crossover control. One example candidate for such a factor is SUMOylated Ecm11,
345
as Ecm11 SUMOylation is required for SC assembly and is impaired in both gmc2 and zip1-N1
346
mutants (HUMPHRYES et al. 2013; Figure 4).
347
Second, the excess MutSγ crossovers observed in ecm11, gmc2 and zip1-N1 mutants may derive
348
from additional recombination events that are initiated and processed specifically during a
349
protracted prophase; such a delay in prophase progression could be caused by a checkpoint
350
triggered by the absence of tripartite SC or SC central element proteins. Indeed, an ndt80 mutation-
351
induced prophase arrest was found to rescue deficiencies in spore viability, synapsis, and
352
interhomolog recombination for some spo11 hypomorphic strains (ROCKMILL et al. 2013), an ndt80
353
mutation-induced prophase arrest was separately found to be associated with elevated
354
recombination initiation in otherwise wild-type cells (ALLERS and LICHTEN 2001; THACKER et al.
355
2014), and elevated interhomolog recombination has been observed in mutants such as zip3, zip1
15
356
and msh5, which exhibit a dual deficit in SC assembly and SC-associated crossing over and have a
357
protracted prophase (THACKER et al. 2014). It is noteworthy that in the case of mutants with a dual
358
deficit in SC assembly and SC-associated crossing over, the extent of elevated interhomolog
359
recombination or recombination initiation could not be fully explained by a protracted prophase
360
alone (ROCKMILL et al. 2013; THACKER et al. 2014), suggesting that either a pro-crossover or an SC
361
assembly activity (or both) can directly modulate interhomolog recombination. Nevertheless, the
362
possibility exists that increased duration in prophase alone, due to a checkpoint response triggered
363
by an SC deficiency, can potentially allow for the accumulation of interhomolog recombination
364
events (including MutSγ-mediated crossovers) in crossover proficient, SC-deficient mutants such as
365
ecm11, gmc2 and zip1-N1.
366
To explore whether an increase in MutSγ crossing over in SC central element-deficient mutants
367
might be due to a prolonged prophase, we examined the morphology of DAPI-stained, surface-
368
spread nuclei and associated spindle structures from wild-type, ecm11, ecm11[K5R, K101R] and
369
zip1-N1 cells in liquid sporulation media at multiple time points (Figure S2A). We also used DAPI
370
staining on whole-mount cells cultured on solid sporulation media to measure the frequency of
371
meiocytes, at multiple time points, that had undergone at least the first meiotic division (Figure
372
S2B). Consistent with a prior study, we found that ecm11 mutants exhibit a delay in exiting meiotic
373
prophase (Figure S2 and HUMPHRYES et al. 2013). In our liquid sporulation time course experiment,
374
by 28 hours approximately 50% of surface-spread nuclei from wild-type meiocytes had progressed
375
beyond the pachytene stage and a substantial fraction were undergoing meiotic divisions. In
376
contrast, DAPI morphology and spindle structures indicated that nearly all ecm11 and ecm11[K5R,
377
K101R] mutant meiocytes were at pachytene at this 28 hour time point (Figure S2A). Similarly, our
378
analysis of meiocytes cultured on solid sporulation media revealed a lag in the accumulation of
16
379
multinucleate cells in ecm11 null, ecm11[K5R, K101R] and gmc2 null mutants relative to wild type
380
(Figure S2B).
381
However, both of our meiotic progression analyses revealed that zip1-N1 mutant meiocytes
382
progress through pachytene and enter meiotic divisions with similar kinetics as wild-type meiocytes
383
(Figure S2A, B), making a meiotic prophase checkpoint less likely to account for zip1-N1’s excess
384
crossover recombination phenotype.
385
Our analysis suggests that the excess MutSγ crossovers observed in ecm11, ecm11[K5R,
386
K101R], gmc2 and zip1-N1 mutants accumulate independent of a protracted prophase. These data
387
may also reveal insight into how recombination intermediates that form during a protracted
388
prophase are processed in budding yeast. It is interesting to note that while ecm11, ecm11[K5R,
389
K101R], gmc2 and zip1-N1 mutants share the same excess crossover phenotype, ecm11,
390
ecm11[K5R, K101R] and gmc2 mutants exhibit a prolonged prophase as well as a set of excess
391
noncrossover interhomolog recombination events that are not present in zip1-N1 mutants (Table
392
S1). These ecm11 and gmc2-specific phenotypes suggest that interhomolog recombination
393
intermediates formed during the protracted prophase of ecm11 and gmc2 mutants may largely be
394
resolved with a noncrossover outcome.
395
396
Conclusion
397
Our data first and foremost demonstrate that tripartite SC is dispensable for the pro-crossover
398
activities of Zip1, Zip2, Zip3, Zip4, Spo16, Msh4 and Msh5 proteins. The ecm11, gmc2, and zip1-
399
N1 mutant phenotypes reveal that two classes of SC proteins exist in budding yeast, one that has
400
both pro-SC and pro-crossover functions, and a second one that is specifically dedicated to SC
401
assembly. This discovery suggests that the pro-crossover function of Zip2, Zip3, Zip4, Spo16 and
402
Zip1 is not based on a role in assembling tripartite SC, but is an independent activity altogether.
17
403
What are the specific roles of these SC-associated proteins in crossover recombination? Our recent
404
observation that an ancestrally-related version of the transverse filament protein, K. lactis Zip1, can
405
rescue crossover recombination in a Zip3, Zip4, Spo16 and Mlh3-dependent but Msh4-independent
406
manner suggests that SC transverse filament proteins have a specialized role in processing
407
recombination intermediates, perhaps even in a manner that parallels or overlaps the activities of
408
MutSγ complexes (VOELKEL-MEIMAN et al. 2015). It will be of particular interest to understand the
409
molecular basis for how budding yeast SC transverse filament proteins promote the maturation of
410
crossover-designated recombination intermediates, and to learn whether the functional relationship
411
between transverse filament proteins and recombination mechanics is conserved in other organisms.
412
Our analysis of ecm11, gmc2 and zip1-N1 mutants also demonstrate that MutSγ-mediated
413
interhomolog crossovers are limited, either directly or indirectly, by the presence of SC central
414
element proteins. These data are not the first to suggest a link between an anti-recombination
415
function and the budding yeast SC (ALLERS and LICHTEN 2001; ROCKMILL et al. 2013; THACKER et
416
al. 2014). However, prior studies that correlated budding yeast SC with a constraint on
417
interhomolog recombination involved mutants missing proteins required for both crossing over and
418
SC assembly, leaving open the question of whether the pro-crossover activity or the SC is key to the
419
mechanism that limits recombination. The data we present here hones in on the SC itself,
420
independent of any pro-crossover activity, as the relevant molecular entity that is linked to limiting
421
MutSγ-mediated interhomolog crossing over. Taken together with recent studies that have observed
422
excess crossing over caused by alterations in the abundance or structure of SC components in C.
423
elegans and rice (LIBUDA et al. 2013; WANG et al. 2015), our data add weight to the idea that a
424
conserved role of the SC structure is to limit interhomolog recombination.
425
426
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Acknowledgements
522
We thank Scott Holmes for comments on the manuscript. A National Institutes of Health Grant to
523
A.J.M, R15GM104827, supported this work.
524
525
Figure Legends
526
Figure 1. ecm11 and gmc2 mutants display excess Msh4-dependent interhomolog crossovers. (A)
527
Proposed arrangement of known structural components of the budding yeast SC (VOELKEL-MEIMAN et
528
al. 2013): Zip1 dimer units (green) orient with N termini oriented toward the midline of the SC central
529
region, where Ecm11 and/or SUMOylated Ecm11 (red) and Gmc2 (gold) assemble to create the SC
530
“central element” substructure. (B) Markers used to define seven genetic intervals in which crossing
531
over was assessed by tetrad analysis. (C) Percentage of wild-type map distance displayed by each
532
strain for each interval (labeled on the x axis). (See Table 1 for raw data (including significance
533
values) and strain names, Table S1 for non-Mendelian (non 2:2) segregation, Table S3 for sporulation
534
efficiency and viability of strains used.)
535
536
Figure 2. ecm11 and gmc2 mutants exhibit robust Zip1-, Zip4- and Msh4- mediated crossing
537
over. A physical assay for crossing over across the entire chromosome III; Southern blotting is used to
538
measure the relative amounts of three forms of chromosome III during a meiotic time course: Aliquots
539
of sporulating cells were taken at 0, 40, and 70 hours after placement in sporulation medium (GAME
540
1992; VOELKEL-MEIMAN et al. 2015). (A) Representative blots show bands that correspond to
541
different sized versions of linear chromosome III present in meiotic extracts from strains indicated
542
above the blot. Circular chromosomes III present in these strains do not enter the gel. The lowest
543
molecular weight band represents linear (“monomer”) III, while the middle and upper bands represent
23
544
crossover products between linear and circular III; the product of a single crossover event runs at the
545
size of the middle band (“dimer”) while a double crossover event involving 3 sister chromatids (of
546
which 2 are circular) produces the upper band, a “trimer” chromatid III. (B) Graph plots three bars (0,
547
40 or 70 hours) for each strain (indicated on the x axis), which each correspond to a % recombination
548
estimate (calculated by summing twice the intensity of the trimer band with the dimer band and
549
dividing the sum by the total intensity of the three bands). See Table S4 for strain names; the data for
550
several controls has been published previously (VOELKEL-MEIMAN et al. 2015).
551
552
Figure 3. zip1-N1 expressing meiotic cells display the same excess of Msh4-dependent
553
interhomolog crossovers observed in ecm11 mutants. (A) The protein encoded by zip1-N1 (TUNG
554
and ROEDER 1998) is depicted below wild-type Zip1. (B) Markers used to define six genetic intervals
555
in which crossing over was assessed (genetic markers differ from the experiment presented in Figure
556
1). (C) Percentage of wild-type map distance displayed by each strain for each interval (labeled on the
557
x axis). (See Table 2 for raw data (including significance values) and strain names, Table S1 for non-
558
Mendelian segregation, Table S3 for sporulation efficiency and viability of strains used). Data for wild
559
type and msh4 was previously reported (VOELKEL-MEIMAN et al. 2015). * The LEU2-THR1 interval is
560
absent from the ecm11 strain.
561
562
Figure 4. Ecm11 fails to assemble coincidently with Zip1-N1 on meiotic chromosomes and
563
Ecm11 SUMOylation is altered in zip1-N1 meiotic cells. (A) Images display surface-spread
564
meiotic prophase stage chromosomes from strains carrying one copy of ECM11-MYC and
565
homozygous for ZIP1 (top row), zip1 (2nd row), or zip1-N1 (bottom 3 rows). Strains are
566
homozygous for an ndt80 null allele, and thus will not progress beyond the pachytene stage of
567
meiotic prophase (XU et al. 1995). Zip1 (green) and Ecm11-MYC (red) assemble extensive,
24
568
coincident linear structures on wild-type meiotic chromosomes (labeled with DAPI; white or blue),
569
but assemble only short stretches and often do not overlap on meiotic chromosomes from zip1-N1
570
strains. Insets in final column show a zoomed region from the corresponding image. Scale, 1 µm.
571
(B) Stacked columns indicate the percentage of nuclei from each strain exhibiting absent or
572
exclusively foci of Zip1 or Ecm11 (“None” or “Dotty”; open), a mixture of Dotty and short linear
573
Zip1 or Ecm11 structures (“Discontinuous”; boxed), or long, linear Zip1 or Ecm11 structures
574
(“Continuous”; solid) on late meiotic prophase chromosomes (n=100-156). (C) Western blot shows
575
unSUMOylated, monoSUMOylated and polySUMOylated forms of Ecm11-MYC from ZIP1, zip1,
576
or zip1-N1 meiotic extracts, prepared as previously described (HUMPHRYES et al. 2013; VOELKEL-
577
MEIMAN et al. 2013). (D) Percentage of mono-SUMOylated (open bar) or poly-SUMOylated
578
(shaded bar) forms of Ecm11-MYC measured at multiple time points for each strain. Error bars
579
represent the range of values from two experiments (the absence of a bar associated with zip1-N1’s
580
polySUMOylated Ecm11-MYC at 26 hours is due to the fact that the same value was obtained in
581
both experiments).
582
583
Figure 5. The fraction of recombination events associated with a crossover does not increase
584
in ecm11 meiotic cells. 3-5 independent cultures of wild type, ecm11, and zip1 strains
585
transheterozygous for the arg4-Nsp, arg4-BglII and the leu2-Cla1, leu2-3,112 heteroalleles were
586
assessed for prototroph formation at ARG4 and LEU2. The values given in the third and seventh
587
column are the average measurement of the fraction of cells that are Arg+ or Leu+ after 3 days of
588
liquid sporulation; (SD)=standard deviation. For each strain, the fraction of Arg+ and Leu+ cells in
589
vegetative cultures at the time of transfer to sporulation medium was also measured; the median of
590
independent replicates for each strain were: (for Arg+) WT: 3.7x10-5; ecm11: 8.1x10-5; zip1: 8x10-5;
591
(for Leu+) WT: 1.9x10-6; ecm11: 1.9x10-6; zip1: 4.9x10-6. The values given in the sporulation
25
592
efficiency column are the percent of sporulated products containing 2, 3 or 4 spores. In the 4th and
593
8th columns is the percentage of all selected heteroallelic recombination events associated with a
594
crossover outcome (crossovers were measured in haploid recombinants, using flanking markers
595
indicated in the cartoon below). Crossover frequency was also assessed in intervals that are
596
unassociated with the selected heteroallelic recombination event (columns 5, 6, 9, 10); for ARG4
597
heteroallelic recombination, crossover frequency was assessed in two intervals on chromosome III;
598
for LEU2 heteroallelic recombination, crossover frequency was assessed in two intervals on
599
chromosome VIII.
600
601
Table 1. ecm11 and gmc2 mutants display an excess of Msh4-dependent meiotic crossover events.
602
Map distances and interference values were calculated using tetrad analysis and coefficient of
603
coincidence measurements as described previously (VOELKEL-MEIMAN et al. 2015). 4-spore viable
604
tetrads with no more than 2 gene conversion (non-2:2) events were included in calculations, although
605
cases where adjacent loci display non-2:2 segregation were considered a single (co-conversion) event.
606
See Table S1 for gene conversion frequencies. Table indicates map distances and their corresponding
607
percentages of the wild-type values for individual intervals, and the map distances and the
608
corresponding percentage of wild type for the entire chromosome (by summing the intervals on III or
609
VIII). The number of chromatids III participating in crossover recombination indicates a general
610
increase in interhomolog events in ecm11 mutants relative to wild type: In wild-type 4-spore viable
611
tetrads (n=512), all of the crossover events on chromosome III in a given tetrad involved two
612
chromatids 45% of the time, three chromatids 26% of the time, and four chromatids 27% of the time.
613
In 4-spore viable tetrads from ecm11 mutants (n=878), all of the crossover events on chromosome III
614
in a given tetrad involved two chromatids only 27% of the time, three chromatids 33% of the time, and
26
615
four chromatids 37% of the time. For the intervals marked with (n.d.), interference measurements are
616
not obtainable using the coefficient of coincidence method due to an absence of NPD tetrads.
617
618
Table 2. zip1-N1 expressing meiotic cells display the same excess of Msh4-dependent
619
interhomolog crossovers observed in ecm11 mutants. Map distances and interference values
620
were calculated using tetrad analysis as described previously (VOELKEL-MEIMAN et al. 2015). 4-
621
spore viable tetrads with no more than 2 gene conversion (non-2:2) events were included in
622
calculations, although cases where adjacent loci display non-2:2 segregation were considered a
623
single (co-conversion) event. See Table S1 for gene conversion frequencies. Table indicates map
624
distances and their corresponding percentages of the wild-type values for individual intervals, and
625
the map distances and the corresponding percentage of wild type for the entire chromosome III (by
626
summing the intervals on III). For the intervals marked with (n.d.), interference measurements are
627
not obtainable due an absence of NPD tetrads. Data for wild-type and msh4 strains was previously
628
reported (VOELKEL-MEIMAN et al. 2015).
629
630
631
632
Supporting Data:
633
Figure S1. Crossover interference is weakened in ecm11 and zip1-N1 mutants, as measured by
634
the “Interference Ratio” method. Cartoons depict robust (solid dark arrow), less robust (solid
635
gray arrow), or undetectable (gray, dotted arrow) interference acting between adjacent genetic
636
intervals on chromosome III and chromosome VIII, as measured by the “interference ratio” method
637
(MALKOVA et al. 2004; VOELKEL-MEIMAN et al. 2015) using the tetrad analysis data associated
638
with Tables 1 and 2. See Table S2 for specific values and Table S2 legend for additional description
27
639
of the method. When 1) the P value associated with comparing the distribution of tetrad types
640
between test intervals and 2) the difference in the calculated map lengths were both found to reflect
641
statistical significance we indicate positive interference (black arrows). Blue arrows indicate a
642
statistically significant interference ratio that is greater than 1. When either the P value or map
643
length difference (but not both) is not statistically significant, or when P values and map difference
644
differences are both significant but interference ratio is two tenths or more higher than the wild-type
645
value for the corresponding interval, interference is considered weakened (solid gray arrow). When
646
both the P value and the differences between map lengths are not statistically significant,
647
interference is either absent (ratios of 0.9 or greater, gray dotted arrow) or inconclusive (gray dotted
648
arrow).
649
650
Figure S2. Meiotic progression in ecm11 and zip1-N1 mutants. Images in (A) display surface-
651
spread nuclei at various time points after sporulating in liquid media for wild type (K842), ecm11
652
(K857), or zip1-N1 (SYC123) strains (genotypes at left of images). Surface-spread nuclei are
653
stained with DAPI to label DNA (white, first column; blue, third column), anti-Red1 antibodies
654
(white, second column; green, third column) and anti-tubulin antibodies (red, third column). Nuclei
655
were classified based on their stage in meiosis (zygotene, pachytene, diplotene, MI or MII) based
656
on the morphology of DAPI-stained DNA, the abundance of Red1 on chromosome axes, and the
657
presence of separated spindle pole bodies or a spindle. Scale, 1 µm. Stacked columns in each graph
658
(below images) indicate the percentage of total meiotic nuclei examined (n=50-75) that are at
659
leptotene/zygotene (open), pachytene (light shaded), diplotene (dark shaded), or are undergoing
660
meiotic divisions or have formed spores (black) at a specific time point. Time points are indicated
661
below the x axis for each strain. Premeiotic nuclei were excluded from this analysis. The graph in
662
(B) shows the percentage of wild-type (K842), ecm11 (K857), ecm11[K5R, K101R] (K846), gmc2
28
663
(K906) or zip1-N1 (SYC123) meiocytes from solid sporulation media that have completed one or
664
both meiotic divisions as a function of time. 100 whole-mount DAPI stained cells were examined
665
for each of three replicates for every strain at every time point. Bars show standard error of the
666
mean.
667
668
Table S1. Non-Mendelian segregation (gene conversion events) per locus, measured in 4-spore
669
viable tetrads. Presented are the percentages of non-Mendelian segregation events (3:1/1:3
670
segregation, top; 4:0/0:4 segregation, below) out of the total tetrads analyzed (second column) in
671
each of the indicated strains. Data is derived from 4-spore viable tetrads with no more than 2 gene
672
conversion (non-2:2) events, although cases where adjacent loci segregate non-2:2 were considered
673
a single conversion event. The sum total percentage of observed non-Mendelian events, and the fold
674
increase relative to wild type, is presented, far right.
675
676
Table S2. Interference ratio data for ecm11, gmc2, and zip1-N1 strains. The Interference ratio
677
values for crossover interference were calculated as previously described (VOELKEL-MEIMAN et al.
678
2015), using the tetrad analysis data associated with Figures 1 and 3, and Tables 1 and 2. Displayed
679
in columns are distribution of tetrad type values, map distances, ratio of map distances, chi-square P
680
value, and significance of differences between map distances for either one or two (flanking)
681
adjacent “test” intervals associated with a “reference” interval (top row). Strain genotypes are
682
indicated at left. See Figure 1 for summary cartoon. The “interference ratio” compares the map
683
distances of an interval when an adjacent interval had, or had not, experienced crossover
684
recombination. To obtain an interference ratio, tetrads that show no evidence of recombination in a
685
“reference” interval (Parental Ditype (PD) tetrads) are sorted from tetrads containing a single or
686
double crossover in the reference interval (Tetratype (TT) and Non-Parental Ditype (NPD) tetrads).
29
687
Next, the distributions of tetrad types and map distances are obtained for an adjacent “test” interval
688
using the tetrads in either reference interval category. The “interference ratio” is derived from the
689
ratio of two map distances associated with the same test interval: the map distance calculated from
690
tetrads in which the adjacent reference interval is recombinant (contains NPD or TTs) divided by
691
the map distance calculated from tetrads that are PD for the reference interval. Since the two map
692
distance values should approximate 1 in the case that a recombination event in an adjacent reference
693
interval has no interfering effect on the frequency of crossing over in a test interval, the interference
694
ratio gives an estimate of the strength of interference; a ratio of less than one can signify positive
695
interference. The significance of differences between map lengths calculated for an interval in
696
either the case of the recombinant or the non-recombinant reference interval was determined using
697
Stahl Online Tools (http://molbio.uoregon.edu/~fstahl/), and a chi-square test (Graphpad.com) was
698
employed to determine if the distribution of tetrad types is considered significantly different in test
699
intervals associated with the recombinant versus the non-recombinant reference interval. When P
700
and the difference in calculated map lengths are both statistically significant, the calculated ratio
701
and these two statistical measurement values are in bold type.
702
703
704
Table S3. Sporulation efficiency and spore viability of strains used for crossover analysis.
705
Sporulation efficiency reflects the fraction of cells that are 2, 3 or 4-spore asci (far right column)
706
after 5 days on sporulation plates. The next to last column shows the spore viability of each strain
707
used in crossover analyses for this study. The frequency of tetrads containing four, three, two, one,
708
or zero viable spores are shown under “Distribution of tetrad types”. (n.d.= not determined.) Full
709
strain genotypes are listed in Table S4. Strains associated with crossover data from Figure 1/Table
710
1 are listed above the set of strains associated with crossover data from Figure 3/Table 2.
30
711
712
Table S4. Strains used in this study. Strains are of the BR1919-8B background (ROCKMILL and
713
ROEDER 1998).
31
MacQueen_Fig.5
Frequency % of Arg+
Frequency % of Leu+
% Crossing
of
of
(n) with
over on chr.III
(n) with
% Crossing over
+
sporulated
sporulated
an
among
Arg
an
on chr.VIII
Sporulation
products associated
products
(n)
associated among Leu+ (n)
Genotype efficiency
that are
that are
crossover
crossover
(n)
Arg+
Leu+
X10-3
X10-4
SPO13- HIS4- CEN3HIS4CEN8- SPO13(SD)
(SD)
THR1
CEN3 MAT
CEN3
SPO13 THR1
ZIP1
(K794)
ecm11Δ
(K829)
zip1Δ
(K826)
54%
(2207)
17.8
(2.2)
70.4
(961)
24
(233)
17
(168)
24.8
(8.0)
47.1
(718)
18
(127)
7
(50)
48%
(1925)
42.2
(2.6)
51.7
(917)
26
(237)
32
(296)
24.4
(6.2)
47.9
(797)
27
(213)
21
(164)
7%
(2074)
92.4
(16.3)
n.d.
43.4
(7.6)
n.d.
n.d.
n.d.
Table 1
GENOTYPE
(STRAIN)
WT
(K842)
msh4Δ
(K852)
mlh3Δ
(K854)
ecm11Δ
(K857)
ecm11[K5R,K101R]
(K846)
gmc2Δ
(K906)
ecm11Δ msh4Δ
(K882)
ecm11Δ mlh3Δ
(K888)
INTERVAL
(CHROMOSOME)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
SPO13-THR1 (VIII)
THR1-LYS2 (VIII)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
SPO13-THR1 (VIII)
THR1-LYS2 (VIII)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
SPO13-THR1 (VIII)
THR1-LYS2 (VIII)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
SPO13-THR1 (VIII)
THR1-LYS2 (VIII)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
SPO13-THR1 (VIII)
THR1-LYS2 (VIII)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
SPO13-THR1 (VIII)
THR1-LYS2 (VIII)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
SPO13-THR1 (VIII)
THR1-LYS2 (VIII)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
SPO13-THR1 (VIII)
THR1-LYS2 (VIII)
PD
TT
344
427
255
395
251
565
296
373
424
275
351
365
423
320
367
415
280
408
325
475
352
456
397
260
314
332
464
210
299
300
174
254
226
338
128
218
210
136
117
183
231
103
358
382
237
292
338
340
244
343
330
207
268
272
324
168
325
250
405
273
401
94
361
96
50
183
115
88
27
129
157
104
232
111
185
41
161
371
426
486
453
441
267
463
316
313
377
324
325
199
364
237
244
278
310
249
178
277
51
28
153
105
56
35
118
109
108
166
129
148
56
179
NPD TOTAL
6
4
14
6
21
1
5
1
1
7
0
3
0
2
0
3
4
0
6
0
2
5
13
29
25
39
8
52
6
10
38
17
32
0
46
7
9
23
15
19
7
28
0
1
5
4
0
0
11
4
2
14
4
8
1
16
675
681
674
674
673
660
662
470
475
465
466
456
450
451
524
522
516
519
516
516
515
832
836
775
792
812
739
725
621
623
589
595
583
537
538
462
463
437
442
451
416
408
409
411
395
401
394
375
373
456
440
387
401
428
381
363
cM
(± SE)
26.7 (1.4)
20.1 (1.2)
36.3 (1.8)
22.9 (1.4)
39.2 (2.0)
7.6 (0.8)
29.5 (1.3)
10.9 (1.1)
5.9 (0.9)
24.2 (1.9)
12.3 (1.0)
11.6 (1.4)
3.0 (0.6)
15.6 (1.4)
15.0 (1.0)
11.7 (1.3)
24.8 (1.5)
10.7 (0.9)
21.4 (1.7)
4.0 (0.6)
16.8 (1.3)
24.1 (1.1)
30.1 (1.5)
42.6 (2.0)
38.1 (1.9)
41.6 (2.2)
21.3 (1.4)
53.5 (2.7)
28.3 (1.5)
29.9 (1.7)
51.4 (2.9)
35.8 (2.1)
44.3 (2.7)
18.5 (1.0)
59.5 (3.3)
30.2 (1.9)
32.2 (2.1)
47.6 (3.1)
45.3 (2.5)
40.2 (2.8)
26.4 (2.1)
54.3 (3.5)
6.2 (0.8)
4.1 (1.0)
23.2 (2.0)
16.1 (1.8)
7.1 (0.9)
4.7 (0.8)
24.7 (2.8)
14.6 (1.6)
13.6 (1.4)
32.2 (2.9)
19.1 (1.8)
22.9 (2.2)
8.1 (1.2)
37.9 (3.2)
%WT
100
100
100
100
100
100
100
41
29
67
54
30
39
53
56
58
68
47
55
53
57
90
150
118
166
106
280
181
106
149
142
156
113
243
202
113
160
131
198
103
347
184
23
20
64
70
18
62
84
55
68
89
83
58
107
128
cM
by chrm
106.0 (III)
%WT NPDobs/NPDexp
by chrm
(± SE)
100
0.19 (0.08)
0.25 (0.13)
0.22 (0.06)
0.30 (0.13)
76.3 (VIII)
100
0.35 (0.08)
0.54 (0.54)
0.11 (0.05)
53.3 (III)
50
0.35 (0.35)
1.41 (1.42)
0.55 (0.21)
n.d
30.2 (VIII)
40
1.22 (0.71)
n.d
0.34 (0.25)
62.2 (III)
59
n.d
1.00 (0.58)
0.20 (0.10)
n.d
42.2 (VIII)
55
0.53 (0.22)
n.d
0.25 (0.18)
134.9 (III)
127
0.16 (0.07)
0.29 (0.08)
0.34 (0.07)
0.41 (0.09)
116.4 (VIII)
153
0.73 (0.13)
0.49 (0.18)
0.60 (0.11)
0.18 (0.07)
145.4 (III)
137
0.31 (0.10)
0.53 (0.11)
0.43 (0.11)
122.3 (VIII)
160
0.77 (0.15)
n.d
0.65 (0.11)
0.27 (0.11)
155.3 (III)
147
0.32 (0.11)
0.45 (0.11)
0.43 (0.04)
120.9 (VIII)
158
0.61 (0.15)
0.50 (0.19)
0.55 (0.09)
49.6 (III)
47
n.d
4.00 (4.02)
0.48 (0.22)
0.95 (0.48)
36.5 (VIII)
48
n.d
n.d
1.82 (0.58)
79.5 (III)
75
1.02 (0.52)
0.50 (0.36)
1.06 (0.30)
0.59 (0.30)
68.9 (VIII)
90
0.93 (0.34)
0.87 (0.88)
0.89 (0.24)
Table 2
GENOTYPE
(STRAIN)
WT
(YT131)
msh4Δ
(AM3313)
ecm11Δ
(AM3378)
zip1-N1
(SYC123)
zip1-N1 ecm11Δ
(SYC142)
zip1-N1 msh4Δ
(SYC151)
zip1-N1 mlh3Δ
(SYC133)
INTERVAL
(CHROMOSOME)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
iTHR1-iLEU2 (XI)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
iTHR1-iLEU2 (XI)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
iTHR1-iLEU2 (XI)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
iTHR1-iLEU2 (XI)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
iTHR1-iLEU2 (XI)
HIS4-CEN3 (III)
CEN3-MAT (III)
MAT-RAD18 (III)
RAD18-HMR (III)
SPO11-SPO13 (VIII)
iTHR1-iLEU2 (XI)
PD
TT
257
340
187
295
219
403
521
526
362
441
465
587
158
159
101
128
115
265
209
215
242
144
417
290
262
302
307
198
432
481
496
407
397
437
503
312
274
246
253
215
371
231
155
288
196
260
90
90
90
230
162
129
33
217
215
232
229
224
303
375
355
329
391
164
459
498
428
427
501
313
116
109
185
199
138
73
157
205
226
221
236
103
NPD TOTAL
8
3
16
5
6
0
3
3
12
7
2
0
4
4
21
7
20
12
17
17
18
40
5
12
18
21
20
50
8
2
2
6
4
2
0
6
12
13
13
20
2
496
498
491
496
485
493
614
619
604
610
596
620
379
378
354
364
362
580
601
587
589
575
586
761
778
751
754
749
753
599
607
598
600
577
576
475
491
485
487
471
476
cM
(± SE)
28.1 (1.9)
17.4 (1.4)
39.1 (2.4)
22.8 (1.7)
30.5 (1.7)
9.1 (0.9)
8.8 (1.1)
8.7 (1.1)
25 (1.9)
16.7 (1.5)
11.8 (1.1)
2.7 (0.5)
31.8 (1.9)
31.6 (1.9)
50.6 (3.5)
37.2 (2.3)
47.9 (3.5)
32.2 (1.9)
39.7 (2.1)
38.9 (2.1)
37.1 (2.2)
54.9 (2.9)
16.6 (1.4)
34.9 (1.5)
39.0 (1.7)
36.9 (1.9)
36.3 (1.8)
53.5 (2.5)
24.0 (1.4)
10.7 (1.1)
10.0 (1.0)
18.5 (1.5)
18.6 (1.3)
13.0 (1.1)
6.3 (0.7)
20.3 (1.8)
28.2 (2.2)
31.3 (2.3)
30.7 (2.3)
37.8 (2.8)
12.1 (1.3)
%WT
100
100
100
100
100
100
31
50
64
73
39
30
113
182
129
163
157
115
228
100
163
181
184
125
224
94
159
175
264
38
57
47
82
43
69
72
162
80
135
124
133
cM
by chrm
107.4 (III)
59.2 (III)
151.3 (III)
147.9 (III)
147.1 (III)
57.8 (III)
110.5 (III)
%WT NPDobs/NPDexp
by chrm
(± SE)
0.38 (0.14)
100
0.39 (0.22)
0.38 (0.11)
0.37 (0.17)
0.20 (0.08)
n.d.
1.64 (0.95)
55
1.65 (0.96)
0.79 (0.24)
1.1 (0.41)
0.49 (0.35)
n.d.
0.14 (0.07)
141
0.14 (0.17)
0.43 (0.12)
0.17 (0.07)
0.50 (0.13)
0.35 (0.11)
138
0.26 (0.07)
0.30 (0.08)
0.42 (0.11)
0.56 (0.08)
0.70 (0.32)
0.17 (0.05)
137
0.19 (0.05)
0.36 (0.09)
0.35 (0.08)
0.43 (0.07)
0.34 (0.12)
0.62 (0.44)
54
0.73 (0.52)
0.65 (0.27)
0.37 (0.19)
0.40 (0.29)
n.d.
0.70 (0.29)
103
0.77 (0.23)
0.63 (0.19)
0.68 (0.20)
0.82 (0.20)
0.61 (0.43)