Strand-specific ChIP-seq at DNA breaks distinguishes single versus doubled stranded DNA binding and refutes single stranded nucleosomes

In a first step of DNA double-strand break (DSB) repair by homologous recombination, DNA ends are resected such that single-stranded DNA (ssDNA) overhangs are generated. ssDNA is specifically bound by RPA and other factors, which constitutes a ssDNA-compartment on damaged chromatin. The molecular organization of this ssDNA- as well as the adjacent dsDNA-compartment is crucial during DSB signaling and repair. However, data regarding the association of the most basic chromatin components – the nucleosomes – have been discrepant. Here, we use site-specific induction of DSBs and chromatin-immunoprecipitation followed by strand-specific sequencing to analyse in vivo binding of key DSB repair and signalling proteins to either the ssDNA- or dsDNA-compartment. In case of nucleosomes, we show that recently proposed ssDNA-nucleosomes are not a major, persistent species, but that nucleosomes eviction and DNA end resection are intrinsically coupled. These results support a model of separated dsDNA-nucleosome- and ssDNA-RPA-compartments during DSB signaling and repair.