SRA

Recent studies suggest that the repair of DNA double-strand breaks (DSBs) is supported by damage-induced small RNAs (diRNAs) that are generated by Drosha and Dicer from transcripts that originate from the regions flanking the break. However, transcription and diRNA production at naturally occurring DSBs in mammalian genomes remain controversial. We have used the homing endonuclease I-PpoI to produce DSBs in human and mouse cells, and we have analyzed the biogenesis of diRNAs at I-PpoI induced breaks in the 28S rDNA and the Ryr2 gene. We provide direct evidence that RNA polymerase II transcribes the sequences that flank the DSBs. Our results also reveal that the resulting transcripts are processed into two types of diRNAs with characteristic length and terminal nucleotide signatures. Furthermore, diRNA production is independent of Drosha and DGCR8, and only a subset of diRNAs depends on Dicer, as shown by small RNA analyses of knock-out cells that lack specific RNA processing enzymes. Our findings are compatible with previous observations of a general transcription inhibition at DSBs and provide novel insights into the mechanisms of RNA synthesis and processing at DSBs. Overall design: Cells (either mESCs or HeLa) were transfected with a plasmid coding for the homing endonuclease I-PpoI that induces a double strand break in the 28S rDNA. After transfection the small RNA fraction was sequenced using standard Illumina sequencing and analyzed for reads mapping to the 28S rDNA.