Extended Data Fig. 6: Affinity purification of different combinations of tagged complexes and comparison of DNA-binding activities.

a, Purification of core complexes that carry combinations of HisFlag (H) and MBP (M) tags on different subunits. All combinations yielded soluble Spo11 (western blot, bottom panel). While the Coomassie-stained gel shown in Fig. 1a suggests that Rec104 may be sub-stoichiometric, the similar relative intensities between MBP-tagged Rec102 and Rec104 in the silver-stained gel (where the MBP tag makes up the majority of each tagged protein’s mass) and anti-MBP western blot indicate that the two subunits have the same stoichiometry (compare lanes 1 with 2, and 6 with 7). The difficulty in purifying soluble Spo11-containing complexes when Rec104 is absent (Extended Data Fig. 1a) further bolsters the inference that the purified core complexes (nearly) always include Rec104. b, Comparison of the DNA-binding activity of core complexes that carried affinity tags on different subunits. All tagged complexes assayed had similar DNA-binding activities.