Drosha-dependent DNA damage response

Fascinating new research led by scientists at the MRC Toxicology Unit (Leicester, UK) explores a key and non-canonical role for Drosha in the DNA Damage Response (DDR). 

In wide-ranging studies Lu et al. show the dependence of DDR early in the response, upstream of the decision to repair double-strand breaks (DSB) using homologous recombination (HR) or non-homologous end-joining (NHEJ), which appear to predominantly occur during G2/M or G1/O phases of the cell cycle respectively, and perhaps reflecting the demand for error-free nature of HR during de novo DNA synthesis or in regions with increased transcriptional activity.

In one cell-based assay, Drosha (and Dicer) were subjected to siRNA knockdown in U2OS cells, which were then subjected to radiomimetic DNA damaging agent bleomycin.  Cells were then probed with Annexin V(-FITC) and DRAQ7™ to determine the proportion of cells in late apoptosis by flow cytometry (BD Biosciences FACSCanto II), which was markedly greater in the cells with knockdown of either Drosha or Dicer.

In such cell-based assays far-red fluorescing viability probe DRAQ7™ shows excellent separation between intact cells and those with compromised membranes.  It has no spectral overlap with Annexin V-FITC, used in this work.  This pairing can be further combined with a mitochondrial membrane potential probe TMRM which can together aid interrogation of the cell death mechanism involved.

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Reference:
Lu, W. T., Hawley, B. R., Skalka, G. L., Baldock, R. A., Smith, E. M., Bader, A. S., ... & Bushell, M. (2018). Drosha drives the formation of DNA: RNA hybrids around DNA break sites to facilitate DNA repair. Nature communications, 9(1), 532.