Fig. 3: Dimeric SPO11–TOP6BL complexes on DNA.
From: Reconstitution of SPO11-dependent double-strand break formation
a–c, AlphaFold 3 model. a, Overview of the entire structure. b, Side view isolating SPO11 and DNA. c, Top view of the DNA. Positions relative to dyad axis are numbered. In b,c, DNA segments coloured darker blue showed biased base composition. A C-terminal α-helix and unstructured segment from TOP6BL26 are omitted for clarity (see Extended Data Fig. 3a,b and Supplementary Discussion 2). d, Inferred contacts between the DNA (mostly sugar-phosphate backbone) and amino acid side chains. Groups of mostly positively charged residues (patches A and B) and active-site residues are shaded; boxed residues are predicted to occupy the minor groove. Bases are numbered relative to the presumed dyad axis. Scissile phosphates are shown in blue. e, Arrangement of catalytically important SPO11 residues in each of the two active sites (coloured as in a). Distances of each Y138 residue from its cognate scissile phosphate are shown. f, Detail view of the positioning helix (**; residues 147–161) from a SPO11 WH domain, with adjacent DNA backbone contacts and the catalytic tyrosine indicated. g, Reconstituting DNA nicking with SPO11 mutants Y138F, E224A and a 1:1 mixture of Y138F and E224A. Top, representative agarose gels of cleavage reactions. Bottom, quantification (mean ± s.d. of n = 3 experiments). h, Supercoil relaxation activity. Reactions containing mixtures of Y138F and E224A SPO11 complexes (as in g) were incubated for the indicated times then deproteinized and separated on agarose gels without (top) or with (bottom) 1 µg ml−1 ethidium bromide (EtBr). The red line highlights the topoisomer ladder that disappears on EtBr-containing gels. The red asterisk highlights a new band on the EtBr gel that corresponds to plasmids that were relaxed covalently closed circles (CCC) after the reaction but that became positively supercoiled upon binding EtBr.
