recombination events, SC subunit turnover may be higher at preferred entry sites but still below the detection level of our assay. Recombination and the Dynamic Architecture of the Budding Yeast SC The pattern of initial post-synapsis Zip1 incorporation near Zip3 foci, together with the fact that SC assembly is contingent upon early steps in the recombination pathway, suggests that recombination-based events establish much of the non-uniform architecture of the SC. One way to think about how the recombination landscape may shape an SC composite architecture is that certain recombination events create a meiotic axis perturbation that similarly interrupts the SC, and at these interruptions SC dynamics are distinct from those in the rest of the structure. Importantly, any model attempting to explain the non-uniform dynamics of SC architecture in terms of recombination events must account for the fact that interhomolog crossover recombi- nation intermediates are not required for establishing such SC architecture. Haploid nuclei that have been genetically ‘‘tricked’’ into entering meiosis can build extensive SC (albeit with a temporal delay), and we observe that initial Zip1-GFP incorpo- ration into the previously assembled SCs in haploid meiotic cells occurs with similar timing and with a qualitatively similar distribution as compared to post-synapsis Zip1-GFP incorporation into diploid SCs. We suggest that initial sites of post-synapsis Zip1 incorporation into haploid SCs reflect a set of recombination intermediates analogous to those that we propose shape SC dynamics in diploids, but which engage the sister chromatid. Interestingly, Zip3-GFP or Zip3-MYC decorates the length of SCs in haploid nuclei (Figure S5), consistent with the idea that an analogous set of SIC-associated recombination events, albeit between sister chromatids, influences the architecture of Zip1 structures that are assembled on haploid meiotic chromosomes. The assembled SC is the context within which at least a subset of meiotic recombination events mature. Moreover, the SC influences the resolution of recombination events. Budding yeast SC-deficient mutants initiate recombination, but the fraction of those double-strand breaks that are repaired to a crossover outcome is diminished [17,18,23,24], and the remaining inter- homolog crossovers exhibited by such mutants do not exhibit interference [16–18,24,38,40]. Interference refers to a nonrandom distribution such that two crossover events rarely occur close together. Interestingly, Fung et al. demonstrated that chromosome axes display a cytological manifestation of interference, in the form of a nonrandom distribution of synapsis initiation complexes, even in the absence of assembled SC [41]. Yet genetic studies on SIC- deficient meiotic mutants suggest that assembled SC is required to generate interfering interhomolog crossovers. These conflicting observations are reconciled by proposing that assembled SC influences the repair outcome of a set of interfering recombination intermediates. As Zip3 sites have been proposed to mark such a set of interfering recombination intermediates, favored sites of post- synapsis Zip1 incorporation into assembled SC (which largely appear near Zip3) may reflect functional interfaces between assembled SC and crossover-designated recombination interme- diates that play a role in maintaining the designation of interfering crossover-destined recombination intermediates and/or play a role in influencing the repair outcome of the associated recombination events. Finally, ongoing SC growth may itself be functionally important for creating a rigid structure that aids in maintaining or influencing the repair outcome of interfering, crossover-destined recombination intermediates. Under this model, our observation SC Dynamics at Steady State that full-length SCs grow continuously in a Zip1 concentration- dependent manner leads to the prediction that meiotic cells carrying a larger Zip1 copy number will maintain an increased capacity to impose interference. Klutstein et al. (2009) recently reported evidence in support of this prediction; these authors discovered that crossover interference is reduced in strains carrying just a single copy of ZIP1 [42]. Methods Strains All diploids are isogenic with BR1919-8B [43]. Strains used in this study are listed in Table S1. Yeast genetic manipulations were carried out via standard procedures. Meiosis-competent haploid strains were constructed using pB211 to integrate MATa at THR1 in a MATa haploid [44]. The ZIP1-GFP fusion construct used for all experiments except for those measuring fluorescence intensity (Figure 6 and Figure 7) is described in [25]. Fluorescence intensity experiments used ZIP1-YFP fusions, in which YFP is inserted between amino acids 700 and 701 of Zip1, as described in [35]. Both fusion constructs were a kind gift of David Kaback. To make strains carrying multiple copies of ZIP1-YFP, a ZIP1-YFP fragment was excised from pRS316-ZIP1-YFP and placed into the LEU2- marked integrating shuttle vector pRS305 [45]. The verified clone, BAM179, was cut with Xcm1 in order to target to leu2. Integration and function of ZIP1-YFP::LEU2 was verified by sporulation rescue of a zip1 and visual assay of fluorescent SCs in live cells. Induction Experiments The TRP1::PGAL1 promoter cassette was placed upstream of the ZIP1 or ZIP1-GFP ORF by directed transformation of a PCR product with homology to the 59 end of ZIP1. pKB80 (GAL4.ER::URA3) was integrated at ura3 to introduce the chimeric protein that responds to b-estradiol and activates PGAL1 promoters [26]. Strains were grown overnight in YPADU media to late log/ early stationary phase at 30u, washed with an equal volume of water and suspended at a 4-fold dilution in 2% potassium acetate (pH 6–6.5). Strains were induced with 1 mm b-estradiol (Sigma E2257, prepared in ethanol). Uninduced cultures were removed from the sporulating culture just prior to induction, and to these cultures an appropriate volume of 95% ethanol was added. 5– 10 ml of sporulating culture was removed at various time points for chromosome spreads or TCA protein preparation. Cytological Analysis and Imaging Meiotic chromosome spreads, staining and imaging were carried out as previously described [46] with the following modifications: 80 ml 1xMES and 200 ml 4% paraformaldehyde fix were added to spheroplasted, washed cells, then 80 ml of resuspended cell solution was put directly onto a frosted slide and cells were distributed over the entire slide using the edge of a coverslip with moderate pressure. The slide was allowed to air dry until less than half of the liquid remained, and then washed in 0.4% Photo-flo as described [46]. The following primary antibodies were used: chicken anti-GFP (1:100) (Abcam), mouse anti c-myc (1:200) (Invitrogen, 9E10.3), affinity purified rabbit anti-Zip1 (1:100) (raised at YenZym Antibodies, LLC, against a C terminal fragment of Zip1 as described in [10]). Secondary antibodies were obtained from Jackson ImmunoResearch and used at a 1:200 dilution. The time course experiments in Figure 7 were carried out over a series of three experiments each containing at least two strains: experiment 1 (which is depicted first of the two datasets for each strain along the X axis in Figure 7) contained all four strains (1 PLOS Genetics | www.plosgenetics.org 14 October 2012 | Volume 8 | Issue 10 | e1002993