Extended Data Fig. 1: DNA fragments released in meiosis are characterized with single-nucleotide precision.
From: Spo11 generates gaps through concerted cuts at sites of topological stress
a, In a rad50S mutant about one-third of the dDSB fragments are robustly resistant to all Exo V concentrations analysed. qPCR of dDSB signals at the YCR047 hotspot (dark blue) or the YCR011 cold region (light blue) using primer pairs PP7 (248 bp) or PP2 (130 bp), respectively (blue bars represent mean values, error bars denote s.d. of three independent experiments, black dots indicate individual values). Digests were performed at 37 °C except for one negative control (4 °C). b, dDSB signals peak during DSB formation in wild-type synchronized meiotic cultures. qPCR as in a (mean and s.d. of two independent experiments are shown, individual values are represented as circles and diamonds). Signals were depleted in the untagged control, the catalytically inactive spo11Y135F mutant and the DSB formation-impaired rec102∆ mutant. c–e, Internal quality controls show single-nucleotide precision and striking signal-to-noise ratio of Protec-seq. c, Diagram explaining the Spo11 signature and the representation of dDSB fragments as arcs. A strong break site produces dDSB fragments in both orientations (grey and green) with a 1-nt offset between Watson (W) and Crick (C) 5′ ends reflecting the 2-nt overhang produced by Spo118 (Spo11 signature). Differently oriented fragments arise from independent cleavage events at different rates. Thus fragments originating at the same break site but ending at opposite sides may show different depths (dDSB asymmetry). d, ‘Cut site fingerprints’ demonstrate single-nucleotide precision in Protec-seq. Cut site fingerprints are single-nucleotide resolution patterns with a window of a few nucleotides around a dominant, isolated Watson signal. Inaccurate determination of 5′ ends would result in a spill over from the dominant signal. Left, averaged fingerprint from 11 cut sites on the Watson strand selected for strength and isolation, with Crick signals serving as independent readout. Watson signals are depicted as filled columns with positive values (percentage of the dominant Watson signal), Crick signals as empty columns with negative values (percentage of the dominant Crick signal). Right, filled and empty columns as in the left panel, but averaged over eight different experiments, involving six different mutant backgrounds at the same cut site (chromosome IV, 824,137). e, Genome-wide detection of the Spo11 signature for assessment of the Protec-seq accuracy. Quantification of the Watson–Crick offset for strong and isolated peaks (Methods) show a high percentage of the 1-nt Watson–Crick offset in the dDSB samples. Wild-type (n = 3,272), tel1∆ rec114-8A (n = 3,726), mre11S (n = 1,155), rad50S (n = 1,947), com1/sae2∆ (n = 1,674), and for comparison Spo11-oligos (n = 1,964, derived from ref. 15) are shown (n denotes the number of peaks that fulfil the selection criteria). f, The architecture of dDSB hotspots is well conserved across resection mutants and the tel1∆ checkpoint mutant. Arcs represent dDSB fragments at a hotspot around 824,100 on chromosome IV, with grey shades corresponding linearly to the fragment depths. Plots in the bottom right show dDSB fragments starting at a single position (116,869, chromosome IV) for wild-type and rad50S strains. Orange bars represent Spo11-oligo 5′ ends. All samples are from t4.
