Strand-specific ChIP-sequencing reveals nucleosome dynamics at DNA double-strand breaks

Abstract

DNA double-strand breaks (DSBs) are highly toxic lesions that, if not correctly repaired, can have detrimental consequences on genome integrity and cell survival. During the course of evolution, different mechanisms developed to repair DSBs including nonhomologous end-joining (NHEJ) and homologous recombination (HR). A pivotal step of the cellular repair pathway decision is the processing of DSB ends during DNA end resection, which, through the degradation of 5’-terminated strands, generates a long stretch of single-stranded DNA (ssDNA) that prevents re-ligation by NHEJ factors and guides repair towards HR. At the same time, resection divides the chromatin surrounding a DSB in distinct ssDNA and dsDNA domains. Establishment of these domains is crucial for HR repair as well as for DNA damage signaling and checkpoint activation, but the protein composition and the interactions between these compartments were not fully understood. Specifically, it was unclear whether nucleosomes, the fundamental unit of chromatin, could be found in the ssDNA domain as well. This would have considerable implications for the recruitment of repair factors to DSBs and for the maintenance of the epigenetic information during repair. However, previously used techniques were inadequate to address this question. Here, we combined site-specific induction of DSBs with chromatin immunoprecipitation, followed by strand-specific library preparation and next-generation sequencing to analyze the in vivo DNA binding mode of key DSB repair proteins as well as nucleosomes. In proof-of-principle experiments, strand-specific ChIP-sequencing recapitulated the characteristic binding pattern of RPA and Rad51 to ssDNA at resected DSBs. Using this technique, we were also able to detect Rad51 binding to dsDNA during homology search. The 9-1-1 signaling platform was suggested to bind at the ss-dsDNA junction at resected DSBs. We showed that, in vivo, 9-1-1 associates with the dsDNA compartment and locates at the leading edge of resection. Furthermore, we did not find evidence of the presence of nucleosomes on ssDNA and, therefore, they do not represent a major species at resected DSBs. In contrast, we found that nucleosomes become fully evicted in concomitance with resection and that the chromatin remodelers RSC and SWI/SNF act redundantly to promote such nucleosome eviction. Taken together, our study revealed that nucleosome eviction is intrinsically coupled with resection and that the ssDNA and dsDNA domains generated by resection are characterized by distinct properties.