Journal: Nature Communications
Article Title: Senataxin resolves RNA:DNA hybrids forming at DNA double-strand breaks to prevent translocations
Figure Lengend Snippet: Senataxin counteracts the formation of translocations. a Rejoining of distant DSBs were detected by PCR, following DSB induction and repair (+4OHT + IAA 2 h) at breaks recently shown to undergo clustering 31 . DNA sequencing confirmed the nature of the amplified products. b MIS12::TRIM37 and LINC00271::LYRM2 rejoining frequencies were analyzed before or after 4OHT + IAA treatment, by quantitative PCR in AID DIvA cells transfected with control or SETX directed siRNA. Mean and s.e.m. of five biological replicates are shown. P values are indicated (one sample t- test). c MIS12::TRIM37 rejoining frequency was analyzed in control or SETX-depleted AID DIvA cells pretreated or not with DRB prior to 4OHT addition as indicated. Mean and s.e.m. of three biological replicates are shown. P value is indicated (paired t -test). d Model: R-loops form as the RNA Polymerase II progresses across the gene. The induction of a DSB elicits ATM activity which triggers RNA polymerase II stalling at the vicinity of the DSB, hence decreasing R-loops across the gene body. On another hand, R-loops and/or RNA:DNA hybrids accumulate in cis to the DSB, due to stalled RNA PolII generating short, abortive, RNAs which thread back in the DNA duplex, or/and potentially de novo PolII transcription from DNA end. Senataxin is further recruited to remove RNA:DNA hybrids at the vicinity of the break induced in active loci. Senataxin and/or R-loop removal, regulate γH2AX establishment, promote Rad51 loading and minimize the occurrence of translocation by a mechanism that still need to be investigated
Article Snippet: RNA extraction and RNA-seq library preparation For RNA-seq, DIvA cells (transfected with the control siRNA) were lysed using TRI reagent (SIGMA) and spiked-in with ERCC RNA Spike-In Mix (Thermo Fisher Scientific).
Techniques: Polymerase Chain Reaction, DNA Sequencing, Amplification, Real-time Polymerase Chain Reaction, Transfection, Activity Assay, Translocation Assay