(A) Intervals analyzed. CEN3 is marked with the ADE2 gene; CEN8 is marked with URA3.
(B) Crossover classes within a single interval and their genetic outcomes.
(C) Contributions of tetratype and non-parental ditype tetrads to map distances in wild-type and sgs1-ΔC795 strains. Asterisks indicate significant differences between map distances in wild-type and sgs1-ΔC795 tetrads (see ; spore viability data are shown in ).
(D) Distribution of crossover classes for the combined intervals along the three chromosomes analyzed. Wild type and sgs1-ΔC795 distributions differ significantly for each chromosome: chromosome 3, P = 0.0008; chromosome 7, P = 3 × l0⁻⁵; chromosome 8, P = 1 × l0⁻⁵.

(A) Map of the HIS4LEU2 locus showing diagnostic restriction sites and the positions of probes. DNA species detected with Probe 4 are shown below. SEI-1 and SEI-2 are the two major SEI species detected with Probe 4 (see ).
(B) Image of one-dimensional (1D) gel hybridized with Probe 4 showing DNA species detailed in (A). Asterisk indicates a meiosis-specific recombinant band resulting from “gene conversion” of the most DSB-proximal XhoI site.
(C) Presumed structures of SEI and dHJ joint molecules.
(D) Image of native/native two-dimensional (2D) gel hybridized with Probe 4. Species detailed in (A) are highlighted. The three dHJ species are highlighted by a trident; SEIs are indicated by a fork.

Row (A): 1D analysis of DSBs and crossovers (COs), and analysis of meiotic divisions (MI±MII). % DNA is percent of total hybridizing DNA. MI±MII is cells that have completed either the first or second meiotic divisions. †, bands resulting from ectopic recombination between HIS4LEU2 and the leu2::hisG allele at the native LEU2 locus (see ). The role of Sgs1 in preventing these events will be described elsewhere (S.D. Oh and N. Hunter, in preparation).
Row (B): 2D analysis of joint molecules in NDT80 cells. In each case a representative 2D panel is shown together with a blowup of the JM region. dHJ species are highlighted by a trident; SEIs are indicated by a fork; large JMs are bracketed.
Row (C): 2D analysis of joint molecules in ndt80Δ cells. “all JMs” = IH-dHJs + IS-dHJs + large JMs.
See also .

(A) Predicted structures, sizes and DNA strand composition of joint molecule species.
(B) Sequential probing of an 8 hr sample from an sgs1-ΔC795 ndt80Δ time-course with common probe, Probe 4, and homolog-specific probes, Probe Dad and Probe Mom (see ). Full panels from native/native 2D gels are shown on the left and blowups are shown on the right. Interpretative cartoon shows the positions of the JM species detailed in (A).
(C) and (D) “Pull-apart” analysis of component strands of the large JMs. (C) Native/native 2D gel highlighting the species of interest. (D) Native/denaturing 2D gel showing that large JMs comprise parental-length component single strands.

(A) Predicted relationships between segment lengths for fully homologous ternary JMs. (B-E) EM images and interpretative cartoons of ternary JMs from an sgs1-ΔC795 ndt80Δ DNA sample taken at 8 hrs: (B) ternary JM comprising three ~4.8 kb molecules interconnected by two closely spaced point junctions; (C) three ~4.8 kb molecules connected by a fused dHJ structure and a point junction; (D) three ~6.9 kb molecules connected by an open dHJ and a fused dHJ; (E) three 6.7 kb molecules connected at a single point, presumably by two very closely spaced point junctions. Segments correspond to those shown in A. Lengths are in kb (see Experimental Procedures). Scale bars = 0.5 μm. See also .

(A) Tetrad analysis of crossing-over in two intervals flanking the HIS4LEU2 locus. Error bars indicate standard errors. See .
(B) Spore viability. At least 200 tetrads were dissected for each strain.
Row (C): 1D analysis of DSBs and crossovers (COs), and analysis of meiotic divisions (MI±MII) from msh5Δ and msh5Δ sgs1Δ-C795 time-course experiments. Data for wild-type and sgs1Δ-C795 cells are from .
Row (D): 2D analysis of joint molecules in msh5Δ and msh5Δ sgs1Δ-C795.
Row (E): 2D analysis of joint molecules in msh5Δ ndt80Δ and msh5Δ sgs1Δ-C795 ndt80Δ cells.

Homologs are shown in red and black, respectively; dashed lines indicate nascent DNA. Solid black arrows indicate pathways in wild-type cells. Dashed arrows indicate pathways in mutants. The crossover or non-crossover decision follows pairing and strand-invasion by one DSB-end to form a nascent D-loop (steps 1 and 2; ; ). Along the crossover pathway, ZMM proteins convert the nascent JM into a SEI, which is then stabilized by Msh4-Msh5 and Mlh1-Mlh3 antagonizing Sgs1 (steps 3B, 4B and 6–8A; also see ). Along the non-crossover pathway, the initial D-loop is not stabilized by ZMMs and ultimately disassembles even in the absence of Sgs1 (steps 3–8D). When homologs have successfully paired and synapsed, the sister-chromatid (or any second homologous template) may be invaded by the second DSB-end, e.g. steps 3D and 4B. Following extension by DNA synthesis, this end undergoes one of two annealing reactions with the first DSB-end. At a crossover-designated site, the second DSB-end anneals with the SEI to form a canonical dHJ, which is then resolved into a crossover (steps 6–8A). At a non-crossover site, the two DSB-ends anneal to seal the break (steps 6–8D). Along both pathways, the helicase activity of Sgs1 (± Top3 strand-passage activity) ensures that the second DSB-end completely dissociates from the template duplex. This could occur early, by disrupting the D-loop intermediate, or late by dissolving dHJs formed by the second DSB-end. In sgs1Δ-C795 cells, the second DSB-end does not efficiently disengage from its template and forms a stable dHJ independently of the first DSB-end, e.g. steps 5C, 6B, 6E. Along the crossover pathway, this will lead to formation of a ternary JM, which may be resolved into adjacent interhomolog (IH) and intersister (IS) crossovers (steps 6–8B). Successive invasion of two templates by a DSB-end will give a quaternary JM, whose resolution can produce a four-chromatid double crossover (steps 5–8C). Along the non-crossover pathway, stable dHJ formation by the second DSB-end will produce an IH-non-crossover associated with an intersister exchange (steps 6–8E). D-loop migration and “end-first” strand-displacement is proposed to be a common step that precedes strand-annealing to form mature JMs. This mechanism readily accommodates the formation of multi-chromatid JMs. Strand-displacement was previously proposed to explain the occurrence of DSB-distal JMs that lack intervening heteroduplex DNA ().