Fig. 4: SPO11 sequence preferences and stimulation of cleavage by dimerization.
From: Reconstitution of SPO11-dependent double-strand break formation
a,b, Sequencing of pUC19 cleavage. Arrow in a indicates the position shown in b (orange dashed line, dyad axis). Read count (average of n = 2 replicates) is in thousands of reads per million mapped reads (KRPM). c, Sequence biases around cleavage sites on pUC19 (i) or genomic DNA from E. coli (ii) or yeast (iii). Read counts at peaks (top to bottom: averaged; as a heat map; sequence logo; sequence colour map (each horizontal line is one peak); and base fractions). RPM, reads per million mapped reads. d,e, Sequence bias in vivo (representative data from one of two experiments). TDP2-seq, tyrosyl DNA phosphodiesterase 2 sequencing. d, Fractional base composition (top) and sequence logo (bottom) are shown around preferred cleavage sites in hotspots43 in Mre11-conditional knockout (cKO) mice35. Note weaker bias compared with c. e, Sequencing reads were averaged across sites matching the in vitro bias (GNATNC, n = 11,423) or the non-preferred reverse sequence (CNTANG, n = 9,018). f,g, Efficient cleavage of an oligonucleotide substrate. f, Schematic of the substrate (details in Methods). g, Cleavage reactions contained 10 nM SPO11 complexes and 0.5 nM DNA. Top, representative autoradiograph with markers for nicks (M-Nick) and DSBs (M-DSB). Bottom, quantification (mean ± s.d. of n = 3 experiments). h,i, Stimulation of DNA cleavage by artificial SPO11 dimerization. Cleavage reactions contained 4 ng µl−1 pUC19 DNA and 50 nM each of FKBP–SPO11–TOP6BL and FRB–SPO11–TOP6BL complexes with or without 5 µM rapamycin. h, Representative gel. i, Quantification (mean ± s.d. of n = 3 experiments).
