Fig. 1: Purification and DNA-binding activity of SPO11–TOP6BL complexes.
From: Reconstitution of SPO11-dependent double-strand break formation
a, Domain organization of dimeric SPO11 complexes and topo VI. Left, side view. Right, top views into the DNA-binding channel, without (top) and with (bottom) DNA. The catalytic tyrosine (Y), metal-binding pocket and hybrid active site (dashed circle) are shown. Figure adapted from ref. 24, CC BY 4.0 (https://creativecommons.org/licenses/by/4.0). b, Coomassie-stained SDS–PAGE of purified Flag–SPO11–TOP6BL preparations (0.5 µg each). Mutant protein purifications were performed at least twice and wild-type purification was performed more than five times with similar results. For gel source data, see Supplementary Fig. 1. M, molecular mass markers; WT, wild type. c, Mass photometry of monomeric SPO11–TOP6BL complexes (28 nM) with or without 5 mM ATP. Particle counts (grey bars), gaussian fits (red lines), fitted mean ± s.d. and percentages of total particles are shown. Asterisks indicate background, which is also present in blanks. d, Top, EMSA of SPO11 complexes binding to a 5′-labelled 25-bp hairpin substrate with a two-nucleotide 5′ overhang end. Bottom, quantification (mean ± s.d. of n = 3 experiments; apparent Kd given as mean ± s.e.m.) is shown. e, DNA length dependence for double-end binding. Top, selected lanes from gel shift assays show binding of SPO11 complexes to DNA of the indicated lengths with two-nucleotide 5′ overhangs on both ends (see full gels in Extended Data Fig. 1d). Bottom, quantification of double-end binding, with yeast Spo11 (ySpo11) data27 for comparison. mSPO11, mouse SPO11. f, AFM analysis of binding to linearized plasmid DNA. Examples are shown of binding to ends (one-end), internally on duplex DNA (duplex), junctions of three DNA arms (three-way) and junctions of four DNA arms (four-way). Percentages of binding particles are shown for n = 200 particles. g, Histogram of bending angles (n = 217 particles bound internally on duplex DNA) compared with randomly chosen protein-free positions (n = 138). Examples are from subpopulations with modal values of approximately 60° and 120°, similar to yeast25.
