Relationships between genome-wide R-loop distribution and classes of recurrent DNA breaks in neural stem/progenitor cells

Recent studies revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes that may give rise to somatic brain mosaicism. The mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. Here, we elucidated the genome-wide landscape of RNA:DNA hybrid structures called “R-loops” in primary neural stem/progenitor cells undergoing aphidicolin-induced, mild replication stress to assess the potential contribution of R-loops to the different, recurrent classes of DNA break “hotspots”. We find that R-loops in neural stem/progenitor cells undergoing mild replication stress are present primarily in early-replicating, transcribed regions and in genes with promoter GC skew that are associated with cell lineage-specific processes. Surprisingly, most long, neural genes that form recurrent DSB clusters do not show R-loop formation under conditions of mild replication stress. Our findings are consistent with a role of R-loop-associated processes in promoter-proximal DNA break formation in highly transcribed, early replicating regions but suggest that R-loops do not drive replication stress-induced, recurrent DSB cluster formation in most long, neural genes.