Extended Data Fig. 1: DNA binding by purified SPO11–TOP6BL complexes.
From: Reconstitution of SPO11-dependent double-strand break formation
a,b, Coomassie-stained SDS-PAGE gel of samples at the indicated steps from a representative purification (a) and representative SEC profile (b). In b, a Coomassie-stained SDS-PAGE gel of the indicated 0.5 ml fractions is above and UV profile (mAU, milli-absorbance units at 280 nm) is below; elution positions of size standards analyzed in a separate calibration run are indicated (arrows); pooled fractions are indicated in red. Purification of wild-type protein was performed more than five times with similar results. c, Representative EMSA of SPO11-Y138F mutant complexes binding to a 25-bp hairpin substrate with a two-nucleotide 5′ overhang end. Quantification is in Fig. 1d. d, DNA length dependence for double-end binding. Full EMSAs are shown for the experiment presented in Fig. 1e. This experiment was performed once. The steric constraints that influence the length dependence of double-end binding are thought to explain the minimum spacing between adjacent DSBs in vivo when multiple Spo11 complexes cut the same DNA molecule in yeast or mouse24,27.
