Extended Figure 8. Progression of meiosis and of recombination in interference-defective mutants.

Representative mutants were examined for progression of meiotic divisions and for recombination at the previously-characterized HIS4LEU2 locus (ref. ⁶⁷; strains in Extended Data Table 1). a, Meiotic divisions. The first meiotic division occurs normally in sir2RK (defective in interaction with Slx5); is delayed in slx5Δ and is completely absent in PCLB2-TOP2 and PCLB-TOP2 top2YF due to arrest at pachytene (ref. ²³; L.Z. unpublished). b, c, DNA events. The HIS4LEU2 locus likely provides a direct readout of DNA events independent of the effects of interference. HIS4LEU2 does not exhibit CO homeostasis²⁴, which implies that is not sensitive to CO interference⁸. This feature presumably reflects the fact that this locus is a very strong DSB hot spot. A DSB occurs at this site in virtually every nucleus with a concomitant reduction in DSBs (and thus CO precursors) at other positions in its vicinity (unpublished results). This locus may also undergo early CO-designation, thus also dominating CO interference patterns per se. Importantly: Zip3 foci are used for diagnosis of CO interference relationships (text; ref. 8). Zip3 foci form as a specific consequence of programmed CO-designation; they do not mark the sites of non-interfering COs, which exhibit an entirely different pattern along the chromosomes⁸. Furthermore, formation of Zip3 foci is upstream of, and thus insensitive to, defects in later events, including: (i) major perturbations in the kinetics of recombination or the fidelity with which initiated events (CO-fated and/or NCO-fated) proceed to their assigned fates (e.g. ref. 14) or (ii) the potential occurrence of additional DSBs due to delayed SC formation (discussion in refs. ⁸ and ⁵⁶). Thus, none of the recombination aberrancies detected by physical analysis of recombination in the analyzed mutants (below) is relevant to their CO interference phenotypes. Correspondingly, while all mutants give exactly the same CO patterns (interference and CO number) as defined by Zip3 foci, the mutants vary widely with respect to DNA recombination phenotypes. The below results can be summarized to say that (i) Absence of Slx5/8-Sir2 STUbL activity has little, or only subtle, effect(s) on recombination whereas (ii) absence of TopoII or TopoII catalytic activity confers delays and aberrancies. b, DSBs, SEIs and dHJs. Progression through recombination is very similar to WT in sir2RK and slx5Δ. Both PCLB2-TOP2 and PCLB-TOP2 top2YF exhibit a phenotype corresponding to delayed progression beyond the point of CO-designation: DSBs appear on time; however, DSBs, SEIs and dHJs all accumulate to higher than normal levels at later than normal times, implying delayed progression of CO-designated DSBs to single-end invasions (SEIs) and of SEIs to double Holliday junctions (dHJs), where SEIs and dHJs are both CO-specific intermediates¹⁴. There is no significant alteration in homolog-versus-sister bias in any of the four mutants, with inter-homolog dHJs predominating over inter-sister dHJs similarly to WT in all cases. c, Inter-homolog crossover (CO) and noncrossover (NCO) products. Inter-homolog CO and NCO levels are very similar to WT in PCLB2-TOP2 and show variations relative to WT in the other mutants. A differential deficit of COs versus NCOs in PCLB2-TOP2 top2YF suggests a specific defect in CO maturation in this mutant.