Drosha drives the formation of DNA:RNA hybrids around DNA break sites to facilitate DNA damage repair. Homo sapiens

The microRNA biogenesis enzyme Drosha was found to be important for DNA repair and this function appears to be distinct to its role in miRNA-mediated repression. Novel small RNAs were reported previously to be produced from the sequences around a DNA break. Utilising an endonuclease system (AsiSI) we were unable to detect such small RNA around 100 cuts within the endogenous genome. Sequencing of R-loops (DNA:RNA hybrids) was performed and an increase in R-loop formation was observed around many DNA break sites. Loss of Drosha appears to perturb this damage dependent formation of R-loops. RNase H1 over-expression appears to reduce repair at these break sites. Drosha appears to be important for facilitating R-loop formation at DNA break sites to aid in the repair process. Overall design: Small RNA sequencing from cells that have been DNA damaged by AsiSI endonuclease at various time points, as well as 5'-triphosphate small RNA (i.e. 5'-monophosphate depleted). 4-thiouridine total RNA PE sequencing to determine transcriptional activity within these cells. DNA:RNA hybrid immunoprecipitation (DRIP) followed by PE sequencing of the genomic fragments, following DNA damage induced by the AsiSI endonuclease with and without siRNA against the microRNA biogenesis enzyme Drosha.