Senataxin (SETX) prevents Myc-induced replicative stress [DRIP-seq]

Here we investigated the RNA helicase Senataxin (SETX), an enzyme that resolve RNA-DNA hybrids and R-loops, to address its role in preventing replicative stress by oncogenic Myc. Silencing of SETX led to selective engagement of the DNA damage response (DDR) upon Myc activation, leading to a robust cytotoxicity. Pharmacological dissection of the upstream kinases regulating the DDR revealed a protective role of the ATR pathway, that once inactivated, boosted SETX driven-DDR. While loss of SETX did not lead to a genome-wide increase of R-loops, mechanistic analyses revealed enhanced R-loops localized at DDR-foci and newly replicated genomic loci, compatible with a selective role of SETX in resolving RNA-DNA hybrids to alleviate Myc-induced RS. U2OS MycER cells (U20S cells expressing the MycER chimera) were infected with virus expressing non-targeting shRNAs (shNT) or shRNA targeting SETX (shSETX). ShRNA expression was activated with 0.2 µg/mL doxycycline, while MycER was activated with 300 nM OHT. Cells were then treated with 100 ng/ml nocodazole (Sigma, Cat. No. SML-1665) for 8h to induce mitotic arrest. G2/M arrested cells were isolated by shake-off, washed with PBS and released in warm medium for 18 hours.