Extended Data Fig. 3: Preference of Spo11 for a DNA-bending motif at periodic distances.
From: Spo11 generates gaps through concerted cuts at sites of topological stress
a, Spo11 cleavage preference and internal GC accumulation in S. cerevisiae as in Fig. 2a, but for 64-nt dDSB fragments (wild-type, t4; n = 164,104, with n being the number of independently identified fragments of a given length). The GC content of 47.8% is lower than for the 34-nt fragments (57.9%), but markedly above the genome average (38.3%). The GC enrichment is not caused by preferential resistance to degradation (data not shown), or by preferential PCR amplification, as the enrichment is robust against deduplication, and GC-rich DNA is known to be underrepresented in over-amplified libraries62. Separate analysis of the 5′ and the 3′ ends of dDSB fragments reveals an asymmetry of base preferences relative to the cleavage axes. The nucleotide preferences inside the fragment (at positions 1, 2 and 13 relative to the cleavage axes) are stronger than their counterparts that flank the dDSB fragment (at positions −1, −2 and −13). All features are robust to deduplication (data not shown). b, c, Cleavage preference and internal GC accumulation in S. kudriavzevii for dDSB fragment lengths of 64 nt (b; 50.4% GC, n = 12,004) and 34 nt (c; 59.4% GC, n = 853) are very similar to their counterparts in S. cerevisiae. Preferential excision of GC-rich sequences could help to limit GC accumulation at yeast hotspots63. d, In tel1∆ rec114-8A double mutants, the periodicity of dDSB fragment lengths was detectable for up to 335 nt. Peak lengths are indicated by pink bars and numbers on the top; valleys are indicated by blue bars. Peaks and valleys were called automatically by a custom algorithm after smoothing with bandwidth = 3 and resolution = 1 (R ‘ksmooth’). e, S. kudriavzevii com1/sae2∆t6 displays a similar distribution of dDSB fragment lengths as S. cerevisiae resection mutants and the same 10.4n + 3 nt periodicity as all S. cerevisiae dDSB samples. f, Quantification of dDSB signal from ref. 15 (gel separation). Left, on the basis of their length distribution, signals are interpreted either as DSB oligonucleotides (dark blue, A1) or dDSB fragments (cyan, A2). Right, diagram illustrating the rationale for estimation of the dDSB fraction F. Each single DSB is represented by two oligonucleotides, one (approximately 30 bp) gives rise to the A1 signal, the other one (14 bp long) is not visible on this gel. dDSB fragments smaller than 70 bp produce two long oligonucleotides with periodic lengths and two with Spo11-oligo lengths that can contribute to the 30-nt peak (A1) (Methods). g, Periodicity does not require nucleosomes. The length periodicity is observed with dDSB fragments lying with both ends in nucleosome-occupied (NN) or in nucleosome-depleted (DD) regions. The nucleosome map is derived from ref. 13 (Methods). h, Table of peak fragment lengths <108 bp averaged from 21 dDSB samples of various genetic background, resulting in a mean peak to peak distance of 10.4 ± 0.36 nt and helix lengths ranging from 11.33 bp (for 34-nt long fragments) to 10.7 bp (for 107 nt).
