Nature. 2021 Jun;594(7864):577-582. doi:10.1038/s41586-021-03632-x
Prieler S, Chen D, Huang L, Mayrhofer E, Zsótér S, Vesely M, Mbogning J, Klein F.
Spo11 generates gaps through concerted cuts at sites of topological stress.
“we developed ‘Protec-seq’ for the purification of end-protected DNA, which includes ChIP of Spo11, ExoV digestion, and the removal of residual 5′-tyrosyl [with hTDP2 (BPS Bioscience)] followed by deep sequencing.”
“dDSB fragments also densely cover chromosomal regions between hotspots, with approximately 31% of cuts outside of hotspots in wild-type cells and around 62% in rad50S mutants”
”The high precision of Protec-seq enabled us to identify that Spo11 has a preference for sequences that partially match a 26-nt long palindromic motif, CN7AAGCA|TGCTTN7G, centred at the cleavage axis (Fig. 2a), and a preference for C and G at position ±13 bp marking the border of the footprint of Spo1133.”
“Dividing the preferred fragment lengths into n helical turns leads to helix lengths above 10.4 bp, indicative of underwound DNA. [...] DNA at promoters is underwound or negatively supercoiled [...] In both wild-type and rad50S cells, (d)DSB levels correlate positively with the corresponding transcriptional stress.”
”As an evolutionary relative of a type IIB topoisomerase, the Spo11 complex may require DNA crossings for the cleavage reaction, which are likely to form at promoters known to accumulate negative supercoils. [...] topoisomerase II (Top2) prefers to bind to DNA crossings under superhelical stress [...] robust [Top2] peaks accumulate at nearly all dDSB sites by the time of DSB formation [...] the occupancy of Top2 increases moderately with higher transcriptional stress at (divergent and tandem) promoters, but not at convergent sites”
“we tested whether gaps that result from dDSBs could account for the observed 6:2 [gene conversion] events by determining the set of dDSB fragments that fully overlap a 6:2 event as an indicator for the local probability of gap formation. The 6:2 event distribution is significantly shifted to genomic positions with higher dDSB gap probability, which indicates that dDSB gaps are enriched at full gene conversion sites and of sufficient length in both wild-type strains and rad50S mutants, as opposed to spo11Y135F mutants”